Techniques in Life Sciences [1 ed.] 9789350438725, 9788184881400

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Techniques •

In

Life Sciences Dr. D. B. TEMBHARE Former Professor and Head,

Department of Zoology And Former Dean, Faculty of Science

Rashtrasant Tukadoji Maharaj Nagpur University Nagpur, India.

K~)1

GRimalaya GFublishing GRouse MUMBAI • DELHI • NAG PUR • BANGALORE • HYDERABAD • CHENNAI • PUNE • LUCKNDW

©

Author

No part of this book shall be reproduced, reprinted or translated for any purpose whatsoever without prior permission of the Publisher in writing.

ISBN: 978-81-84881-40-0 First Edition

Published by

2008

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Contents Protocols

(i - ix)

1

(1-26)

2.

Introduction

1. Cell and Biodiversity 2. Tissue Concept 3. Membranes

9 18

4. Staining Reactions

22

Elements of Microtomy

1. Pre microtomy Processes 2. Microtomy Process 3. Post-microtomy Process

3.

In Situ and Histological Staining Techniques 1. Whole Mount (In Situ) Staining Techniques

2. Histological Staining Techniques

4.

(27-46) 27 40 45 (47-78) 47 53

Cytological and Microbial Staining Techniques - (79-94)

1. Cytological Staining Techniques 2. Microbial Staining Techniques 5.

1

Histochemistry

1. General Histochemistry 2. Enzyme Histochemistry 3. Immunohistochemistry

79 86 (95-122) 95 106 119

6.

Microscopy 1. Light Microscopy 2. Electron Microscopy 3. Three Dimensional Microscopy

7.

8.

Haematological Techniques 1. Blood Composition 2. Haematological Techniques Biochemical Methods 1. Centrifugation / Sedimentation 2. Photometry / Spectroscopy 3. Chromatography 4. Electrophoresis

9.

10.

130 135

(137-154) 137 142 (155 - 186) 155 160 168 177

Detection of Carbohydrates 1. Chemistry and Classification 2. Qualitative Detection 3. Quantitative Detection

(187 - 208)

Detection of Lipids

(209 - 226)

1. Chemistry and Classification 2. Qualitative Detection 3. Quantitative Detection

11.

(123-136) 123

Detection of Amino Acids and Proteins 1. Amino Acids 1.1. Chemistry and Classification 1.2. Quantitative Detection 2. Proteins

187 190 193

209 213 214 (227-254) 227 227 231 234

2.1. Chemistry and Classification

234

2.2. Qualitative Detection 2.3 Quantitative Detection

240 241

12.

Detection of Enzymes 1. Chemistry and Classification 2. Qualitative Detection 3. Quantitative Detection

13.

Detection of Excretory Substances

1. Chemistry 2. Qualitative Detection 3. Quantitative Detection

(255-282) 255 259 261 (283-304) 283 286 290

Detection of Vitamins 1. Chemistry and Classification 2. Quantitative Detection

(305-318) 305

Nucleic Acid Biotechniques 1. Salient Features 2. Laboratory Biotechniques

(319-354) 319

16.

Immunological Techniques 1. Elements of Immunology 2. Immune Reactions 3. Immunological Techniques

(355-374) 355 358 361

17.

Radio-immunoassay of Hormones

(375-400)

14.

15.

1. Principle of Radio-Immuno Assay 2. Chemistry and Classification of Hormones 3. Radioimmunoassay (RIA) Techniques for Hormones -

18.

Animal cell and Tissue Culture 1. Salient Features 2. Cell Culture techniques 3. Cell Culture and Immunocytochemistry

309

328

375 376 384 (401-430) 401 409 423

References

(431-442)

Appendix

(443-458)

Subject Index

(459-463)

Protocols A. CELL AND TISSUE STAINING TECHNIQUES I.

II

Whole Mount (In situ) Staining Techniques l. Grenacher's Alcoholic Borax Carmine Staining Technique 2. Mayer's Carmalum Technique 3. Borror's Whole Mounting Method For Protozoa 4. Kessel and Chen's Whole Mount Method For Intestinal Protozoa 5. Whole Mount Stained Preparation of Chick Embryo 6. Whiting Whole Mounting Method for Ovaries 7. Gurr's Whole Mount Method for Nervous System 8. Zawarzin's Intra Vitum Technique for Nervous System 9. Altman And Bell's Technique For Neuron Cells in Ganglia 10. Hagmann's Trypan Blue Staining Technique for Tracheae

47 48 48 49 50 51 51 51 52 52

Histological Staining Techniques l. Haematoxy lin - Eosin 2. Iron Haematoxylin - Orange G 3. Haemalum - Eosin - Orange G 4. Mallory's Triple 5. Mallory - Heidenhain 's Azan - Aniline Blue 6. Masson's Trichrome 7. Milligen's Trichrome 8. Weigert - Van Gieson's Staining Technique for Connective Tissue 9. Romeis's Bone Staining Technique 10. Younpeter's Alizarin Red S Technique for Skeletal System 11. Maximov's Eosin-azure Bone Marrow Staining Technique 12. Bodian Protargol Staining Technique for Neuroanatomy 13. Holms-blest's Silver Impregnations Technique for Nerve Fibers 14. Hutchison's Berlin Blue Staining Technique for Hemosiderin 15. Glenner's Bilirubin Staining Technique

53 54 56 57 57 58 59 60 60 61 62 62 63 65 65

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. III

IV

Bargmann's Chrome-alum Haematoxylin Phloxine (CHP) Technique Cameron and Steel's Aldehyde Fuchsin (AF) Technique Ewen,s Aldehyde Fuchsin (AF) Technique Adam And Sloper's PerformicAcid -Alcian Blue (PAAB) Technique Herlant's Alcian Blue Phloxine (ABP) Technique Panov's Para-aldehyde Thionine Phloxine (PTP) Technique Heidenhain's Azan Aniline Blue (AAB) Technique Herlant's Alcian Blue Pas (AB-PAS) Technique Mac Conaills-solcia's Lead Haematoxylin Technique DograAnd Tondon's PerformicAcid Victoria Blue (PAVB) Technique Dogra And Tondon's Performic Acid Aldehyde Fuchsin (PAAF) Technique Ittycheriah's Performic Acid Resorcin Fuchsin (pARF) Technique Humberstone's PerformicAcid Victoria Blue (PAVB) Technique Sterba's Pseudoisocynin (PIC) Fluorescent Microscopic Technique Fontanalgomori's Methenamine Silver Technique Lillie And Burtner's Ferric-ferricyanide Technique

66 66 67 68 69 70 71 71 73 73 74 75 75 76 77

78

Cytological Staining Techniques 1. Ludford's Osmium Tetraoxide Technique for Golgi 2. Elftman's Direct Silver Technique for Golgi 3. Altmann's Aniline Fuchsin Technique for Mitochondria 4. Novelli's Acid Fuchsin Technique for Mitochondria 5. Neutral Red - Janus Green B Supravital Technique 6. Acetocarmine Staining of Salivary Gland Chromosomes 7. Giamsa Staining for Chromosome Banding Pattern 8. Goh's Culture Technique for Mitotic Chromosomes 9. Squash Technique for Meiotic Chromosomes to. Klinger And Hammond's Technique for Sex Chromatin 11. Moore's Cresyl Fast Violet Method for Sex Chromatin 12. Puchtler's Congo Red Technique for Amyloid 13. Chrysoidin Staining Technique for Mast Cells

79 80 80 81 81 82 82 83 84 84 85 85 86

Microbial Staining Techniques 1. Gram-weigert's Paraffin Section Technique for Bacteria 2. Leaver's Paraffin Section Technique for Bacteria 3. Gram's Culture-smear Technique for Bacteria 4. Negative Staining Technique for Bacterial Capsule 5. Hiss's Staining Technique for Bacterial Capsule 6. Schuffer And Fulton's Staining Technique for Bacterial Spores 7. Ziehl-nielsen's Acid Fast Staining Technique for Pathogenic Bacilli 8. Ziehl-nielsen's Sputum Smear Technique for Thberculosis Bacilli

86 87 88 89 89 89 89 90

(ii)

9. 10. 11. 12. 13. 14. 15. 16. 17. V

VI.

Ziehl-nielsen's Skin Smear Technique for Leprosy Bacilli Albert's Staining Technique for Diphtheria Bacilli Fontana's Silver Salt Staining Technique for Spirochetes Jenner-giemsa Stain for Malaria Parasite Alpha's Giamsa Staining for Rickettsiae and Psittacossis Vib Gomori's Staining for Trachoma Viral Inclusion Bodies (vib) Schleifstein's Staining Technique for Negri Bodies Hotchkiss- mcmanus Staining Technique for Fungi Gridley's Staining Technique for Fungi

90 91 91 92 92 93 93 93 94

General Histochemistry 1. Kurnick's Methyl Green - Pyronin Y for Nucleic Acids 2. Feulgen Reaction for Deoxyribonucleic Acid (DNA) 3. Toluidine Blue Technique for Ribonucleic Acid (RNA) 4. Chapman's Mercury Bromophenol Blue Technique for Proteins 5. Yasuma and Ichikawa's Ninhydrin - Schiff Reaction for Proteins 6. Sakaguchi's Technique for Arginine-rich Proteins Alfert and Geschwind's Technique for Histone - Proteins 7. 8. Millon's Reaction for Tyrosin - rich Proteins 9. Ferric-ferricyanide Technique for SH - Proteins 10. McManus Periodic Acid Schiff's Reaction for Carbohydrates 11. Best's Carmine Technique for Glycogen 12. Bauer's Feulgen Reaction for Glycogen 13. Putt's Alcian Blue Technique for Acid Mucopolysaccharides 14. Chiffelle & Putt's Sudan Black B or Sudan IV Tech. for Lipids 15. Berenbaum's Acetone - Sudan Black B Tech. for Bound Lipids 16. Elfmann's Sudan Black B Technique for Phospholipids 17. Baker - Hori' s Acid Hematein Technique for Phospholipids 18. Cain's Nile Blue Technique for Neutral and Acidic Lipids 19. Adam's Os04-a-Naphthylamine Tech. for Cholesterol and Lipids 20. Grundland et al:S Digitonin Reaction for Cholesterol 21. Wichard and Komnick's Tech. for Chloride in Respiratory Epithelia 22. Von Kossa's Silver nitrate Technique for Calcium 23. Puchtler's Alizarin Technique for Calcium 24. Hillarp and Hokfelt's Modified Technique for Adrenalin and Noradrenalin 25. Puchtler's modified Technique for Haemoglobin and Haemosiderin

95 96 96 97 97 98 98 98 99 99 100 101 101 102 102 102 103 103 103 104 104 104 105 105 106

Enzyme Histochemistry 1 Gomori's Calcium-Cobalt Technique for Alkaline Phosphatase 2 Fredricson's Calcium - Cobalt Technique for Alkaline Phosphatase 3 Gomori's Lead Nitrate Technique for Acid Phosphatase

107 108 109

(iii)

4 5 6. 7 8. 9. 10. 11. 12. 13. 14. 15. 16 17 18 19. 20.

Takauchi and Tanoue's Technique fo1' Acid Phosphatase Lake's Lead Acetate Technique for Acid Phosphatase Burstone and Folk's Technique for (Proteolytic) Aminopeptidase Moloney et ai's Technique for (nonspecific) Esterases George and Ambadkar's Technique for Lipase Chiquoine's Technique for Glucose - 6 - Phosphatase Wachstein and Meisel's Technique for Glucose - 6 - Phosphatase Burstone's NBT Technique for Succinic Dehydrogenase Cohen's NBT Technique for Glucose - 6 - Phosphate Dehydrogenase Cohen's NBT Technique for (DNPHfTNPH - Diphorase) Bara's NBT Technique for Lactic Dehydrogenase (LD) Laidlaw's Dioxyphenylalanine Technique for Dopa Oxidase Bara's (Wattenberg's modified) NBT technique for ~5 3~-HSD Urano's NBT Technique for Monoamine Oxidase Karnovsky' Technique for Acetylcholinesterase Padykala and Herman's Technique for Adenosine triphosphatase Eckert and Boschek's Technique for Horseradish peroxidease

VII. Immunohistochemistry 1. Indirect Peroxidase-labelled Antibody Technique 2. Immunoperoxidase Method for Auto-antibodies 3. Streptavidin-Biotin-Peroxidase (SBP) Immunohistochemical Technique 4. Double Immunolabeling (Immunohistochemical staning) Technique

109 110 110 111 111 112 112 112 113 113 114 114 115 117 117 118 118

119 120 120 122

B. PHYSIOLOGICAL AND BIOCHEMICAL TECHNIQUES VIII. Haematological Techniques 1. Total Erythrocyte Count (TEC) 2. Total Leucocyte Count (TLC) 3. Differential Leucocyte Count (DLC) 4. Macro hematocrit Technique for Determination of Hematocrit 5. Determination of Total Platelet Count (TPC) 6. Determination of Total Reticulocyte Count (TRC) 7. Determination of Total Eosinophil Count (TEC) 8. Acid hematin Technique for Determination of Hemoglobin 9. Preparation of Hemin Crystals 10. Duke's Technique for Determination of Bleeding time 11. Capillary Method for Determination of Blood Clotting Time 12. Quick's Method for Determination of Prothrombin Time 13. Determination of Thrombin Time 14. Kjeldahl-Nesslerization Method for Determination of Fibrinogen

(iv)

142 144 145 147 147 149 149 150 151 152 152 153 153 153

IX.

X.

Detection of Carbohydrates l. Molisch's Test 2 Iodine Test Fehling's Test 3 4 Benedict's Test 5 Picric Acid Test Barfoed's Test 6 7 Seliwanoff's Test Anthrone Technique for Total Carbohydrates 8 Phenol Sulphuric Acid Technique for Total Carbohydrates 9 10 Nelson-Somogyi Technique for Reducing Sugars 11 Dinitrosalicylic Acid Technique for Reducing Sugars 12 Glucose Oxidase Technique for Glucose in Plant Extract 13 Ortho-toluidine Mono-step Technique for Plasma/Serum Glucose 14 Glucose Oxidase Enzymatic Technique for Blood Glucose 15 Glucose determination by Discrete Autoanalyzer 16 Hexokinase (Rate of Reaction) Tech. for Plasma Glucose 17 Titration Method for Urine Glucose 18 Anthrone reaction for Tissue Glycogen 19 Anthrone Technique for Starch 20 Juliano's Iodine Technique for Amylose 21 Updegroff's Anthrone Technique for Cellulose 22 Goering and Vansoest's Technique for Hemicellulose 23 Ashwell's Resorcinol Technique for Fructose 24 Dahlman's Enzymatic Technique for insect blood sugar, Trehalose 24 Separation of carbohydrates by Paper Chromatography 25 Thin Layer Chromatographical Analysis of Sugars in Urine

191 191 191 191 191 192 192 193 194 194 195 196 196 198 199 200 200 201 202 203 204 204 205 206 207 208

Detection of Lipids l. Sudan III Test for Lipids 2. Acrolein Test 3. Rancid oil Test 4. Copper sulphate Test for Glycerol 5. Liebermann - Burchard Reaction for Cholesterol 6 Sulpho-phosphovanillin Technique for Total Lipids 7 Fossati and Principle's Enzymatic method for Serum Triglycerides 8 Watson's Colorimetric Technique for Serum Total Cholesterol 9 Watson's Colorimetric Technique for Serum HDL Cholesterol 10 Roschlau et at's Enzymatic Technique for Cholesterol by Autoanalyzer 11 Wybenga & Pileggi's method for Cholesterol 12 Stoddart and Drury's Technique for Total Fatty Acids in Blood

213 213 213 213 214 214 215 216 217 218 219 220

(v)

13 14 15 16 17 18

XI.

XII

Stern and Shapiro's Technique for Esterified Fatty Acids in Blood Cox and Pearson's Technique for Free Fatty Acids Titration Technique for Saponification Value Connerty, Briggs and Eaton's Technique for Serum Phospholipids Gomori's Technique for Inorganic Phosphorus in Serum and Plasma Thin Layer Chromatography (TLC) of Neutral Lipids

222 223 223 224 225 226

Detection of Amino acids Ninhydrin Reaction Method for Amino Acids from Urine 1 2 Circular Paper Chromatography of Amino Acids Thin Layer Chromatography (TLC) of Amino Acids 3 4 Estimation of Tryptophan in Proteins Ferric Chloride Reaction for Free Tryptophan Amino Acid 5

231 232 232 233 234

Detection of Proteins 1 Biuret Reaction 2 Ninhydrin Reaction 3. Millon's Reaction 4. Xanthoprotein Test 5. Precipitation Test 6. Hopkins-Cole's Reaction 7 Lowry et a/'s Biuret Technique for Total Proteins 8. Bradford's Technique for Total Proteins Bronsocresol Green (BCG) Technique for Albumin 9 10 Peroxidase Method for Plasma Haemoglobin 11 Dimethyl Sulfoxide (DMSO) Technique for Total Serum Bilirubin 12. Paper Chromatography of Serum Proteins 13 Laemmli's Technique of SDS-PAGE for Separation of Proteins 14 Silver staining technique for SDS-PAGE proteins 15 Radola and Graesslin's Isoelectric Focusing Technique 16 Turbidimetry Method for CSF (Cerebrospinal fluid) and Urinary Proteins

240 240 240 240 240 241 241 243 244 246 247 248 249 252 252 254

XIII Detection of Enzymes 1 Amylase Enzyme Activity Pepsin Enzyme Activity 2 3 Trypsin Enzyme Activity 4 Lipase Enzyme Activity 5 Milk Aldehyde Dehydrogenase Enzyme Activity 6 The p-nitrophenyl Phosphate Technique for Alkaline Phosphatase 7. King and Armstrong's Method for Alkaline Phosphatase 8. King's p-NitrophenylphosphateTechnique for Acid phosphatase

(vi)

259 259 260 260 261 261 262 263

9 10 11 12 13 14 15 16 17 18 19 20 21 22 2324 25 26

Gutman and Gutman's Technique for Acid Phosphatase Yamaguchi et ai., and Le Bel et ai., Technique for ATPases Nachlas et ai., Technique for Succinic Dehydrogenase (SDH) King's Technique for Tissue Lactate Dehydrogenase (LDH) Micromethod for Serum Lactate Dehydrogenase (LDH) Kornberg and Horecker's Technique for G-6-PD King's Technique for Glucose-6-Phosphatase (G -6-Pase) Biggs, Carrey and Morrison's Technique for Acetyl Cholinesterase Wolfgong Pilz's Technique for Acetyl Cholinesterase Bernfield's Dinitrosalicylic Acid Technique for Amylase Nelson-Somogyi's modified Technique for Amylase Ishaaya and Swirski Technique for Amylase Titration Technique for Determination of Amylase in Duodenal Contents Cherry and Crandall's Titration Technique for Lipase Snell and Snell's Technique for Protease The NADH Technique for GOT or AST Bergmeyer and Hordes Technique for GPT or ALT Huge's Modified Technique for Creatine Phosphokinase (CPK)

264 265 266 267 268 269 270 271 272 273 274 275 276 278 279 280 281 281

XIV Detection of Excretory Substances 1.

2 3 4. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Urease Test for Urea Oxidation or Murexide Test for Uric Acid Urease Test for Ammonia Jaffe's Test for Creatine Benedict's Test for Glucose Osazone Test for Lactose Seliwanoff's Test for Fructose Sulphosalicylic acid Reaction for Protein Rothera's Test for Ketones Hay's Sulphur flower Test for Bile Salts Harrison Spot Test for Bile Pigments and Urobilinogen Peroxidase Test for Occult Blood in Urine Jaffe's Reaction for Serum Creatinine DMA Uric Acid Uricase Technique for Serum Uric Acid Glutamate Dehydrogenase Technique for Blood Urea Nitrogen Folin Method for Amino Acid Nitrogen in Blood Flame Photometrical Estimation of Sodium (Na) and Potassium Mercuric Thiocyanate Method for Serum Chloride Schales and Schales Titration Method for Chloride Titrimetric Method for Serum Bicarbonate Daly and Ertingshausen's Molybdate Method for Serum Phosphorous

(vii)

286 286 287 287 287 288 288 288 289 289 289 290 290 291 292 293 294 297 298 298 299

22 22 23 24 25 26 XV

O-Cresolpthalein Complexon Method for Serum Calcium Baron and Bell's Titration Technique for Calcium Ramsay's Dipyridyl Technique for Serum Iron Bathophenanthrolin Technique for Iron Sodium Diethyldithiocarbamate Technique for Copper Titan yellow colorimetric Technique for Magnesium

Detection of Vitamins 1 Peterson and Wiggis Technique for Vitamin A 2 Gloster and Harris Enzymatic Technique for Thiamin (Vitamin B) 3. Fluorimetric Technique for Thiamin (Vitamin - B ,) 4. Fluorimetric Technique for Riboflavin (Vitamin - B2 ) 5. Colorimetric Technique for Niacin (Vitamin - B3) 6. Fluorimetric Technique for Pyridoxine (Vitamin-B 6) 7. Enzymatic Assay of the Vitamin, Folic Acid 8. Titration Technique for Ascorbic Acid, Vitamin C.

300 300 301 302 303 304

309 310

311 312 313 314 316 317

C. RECENT TRENDS IN BIOTECHNIQUES XVI Nucleic Acid Biotechniques 1. Burton's Diphenylamine Technique for DNA 2. Disch's Orcinol Technique for RNA 3. Cultivation of Lambda (A.) Bacteriophage 4. Extraction Technique of Lambda (A.) DNA 5. Alkaline Lysis Technique for Isolation of E. coli Genomic DNA 6 Isolation of Plasmids from E coli Bacteria Cells 7. Alklaline Lysis Technique for Isolation of Plasmid DNA from E. coli 8. Dellaporta, Wood and Hick's Total (Plant) DNA Extraction 9. Phenol-SDS Technique for Total (Plant) RNA Extraction 10. Rapid Technique for Isolation of RNA from Animal Tissue 11. Agarose Gel Electrophoresis Technique for DNA 12. Southern Blotting and Hybridization Technique 13. Polymerase Chain Reaction (PCR) 14 The In Situ PCR: Amplification and Detection in a Cellular Context 15. Gene Cloning Technique 16. Western Blotting Technique (Capillary Blotting Method)

328 329 330 331 332 333 336 337 338 339 340 341 344 346 350 353

XVII Immunological Techniques 1 Antigen-Antibody Slide Method for Blood Grouping 2. Antigen-Antibody Tube Method for Blood Grouping 3. Antigen-antibody Tube Method for Rho (D) Type Blood Grouping 4. Immunological Test for Human Chorionic Gonadotropin (HCG) 5 Rocket Immunoelectrophoresis

361 362 362 363 364

(viii)

6 7. 8 9 10 11 12

367 367 369 370 371

Mancini's Radial Immunodiffusion Method Ouchterlony Double Immunodiffusion Test Isolation of Antibodies, Immunoglobulins (IgG) ELISA by Double antibody Sandwich Technique Cellular ELISA Technique Flow Cytometric Determination of Leukocyte Surfa~e Antigens Direct Immunofluorescence Labeling

372

373

XVIII Radioimmunoassay (RIA) Techniques 1 Bioassay of Triiodothyronine (T3) Hormone 2 Bioassay of T4 Thyroxine (3, 5, 3' 5' - Tetraiodothyronine) Hormone 3. Bioassay of Thyroid Stimulating Hormone (TSH) 4 Bioassay of Follicl,e Stimulating Hormone (FSH) 5. Bioassay of Luteinizing Hormone (LH) 6. Separation of LH from other Proteins on a CM Sephadex Gel Column 7. Bioassay of Prolactin (PRL) by Sandwich ELISA Technique 8 Bioassay of Estradiol 9 Bioassay of Progesterone 10 Bioassay of 17 ex. -OH Progesterone

385 387 388 389 390 392 392 394 395 397

XIX Cell Culture Techniques 1. Standard Cellrrissue Technique 2. Subculture of Adherent Cell Lines (Monolayer Cell Lines) 3. Subculture of Semi-adherent Cell Lines 4. Subculture of Suspension Cell Lines 5. Trypan Blue Dye Exclusion Method for Cell Quantification 6. MTT (Yellow Tetrazolium Salt) Cell Proliferation Assay 7. Cryo-preservation of Cell Lines 8. Lyophilisation and Freeze - Dry Technique for CellfTissue Preservation 9. Revival of Resuscitation of Frozen Cell Lines 10. Detection of Bacterial and Fungal Contamination 11. Detection of Mycoplasma contamination by Hoechst Staining Technique 12 Harvesting and Plating Bone Marrow-Derived Microphages 13 Isolation and Growth of Mouse Primary Myoblasts

409 410 411 412 413 414 415 416 416 417 418 419 420

xx

Cell Culture and Immunocytochemistry 1. Light Microscopic Immunocytochemistry of the Cells in Tisf,ue Culture 2 Quick Immunostaining Method for Cells in Culture 3 Double Antibody Conjugation Method of Immunocytochemistry Annexure Immunohistochemi'stry on Fixed Paraffin-embedded Sections

423 425 426 427

••• (ix)

INTRODUCTION

1.

CELL AND BIODIVERSITY

1.1

Cell Theory

M. Malpighi (1628 -1694) was, however, the first biologist who had studied the thin sections of animal tissues of the brain, liver, kidney, spleen, lungs and tongue and proposed a hypothesis that all the tissues of an animal body are composed of almost alike structural units, the "utricles." Robert Hooke (1665) replaced the term "utricles" by the "cells," means, the hollow spaces alike the honey comb cells as he observed under his own designed compound microscope. In this direction, the great biologist, Leeuwenhoek (1675) deserves, undoubtedly, the first credit to use lenses of the magnification up to 300 X and to observe the microscopic organisms like bacteria, Spirilla, protozoans, rotifers and Hydra in soil and water samples quite distinctly. He was the first biologist to describe spermatozoa of men, dogs, rabbits, frogs, fishes and insects and blood cells of mammals, birds, amphibians and fishes. Later on, Robert Brown (1831-33) discovered the nuclei at the center of the cells of epidermis, stigmas and pollen grains of the plant, Tradescantia. On the basis of consistent microscopic findings of Oken (1805), Mirbel (1807), Lamarck (1809), Dutrochet (1824), Turpin (1826) and Robert Brown (1831), the fascinating doctrine of "cell theory" was proposed by Schleiden and Schwann (1835-39) that "the cell filled with a mass oJprotoplasm (a nucleus embedded in a cytoplasm) represents afundamental structural andfunctional unit of the plant and animal life." Later on, Nageli's (1855) principle of cellular basis of life's continuity and substantial experimental evidences put forth by Louis Pasteur (1865) strongly suppot:ted the cell theory. The cell theory states that1.

Cell is a smallest basic unit of structure and function in all life;

2.

Body of the living organisms universally represents either an individual cell (unicellular organisms) or aggregations of cells and tlieir products (multicellular organisms);

Techniques in Life Sciences

2

3.

All metabolic processes occurring in unicellular and multicellular animals, primarily take place in the individual cells;

4.

New cells (offsprings) originate from the pre-existing or old (parental) cells through division or duplication process and never from de novo spontaneously.

With the advancement of research during the nineteenth and twentieth century, the cell theory which received initially, universal acceptance has been extended to its full dimension that "the cell is the structural and functional unit of the living matter and is capable of carrying on the processes of life independently". The living matter is, in fact, the protoplasm and it is well-equipped with a biological machinery capable of performing all most all activities that an animal conducts in a day to day life. The protoplasm was further divided into two parts, nucleoplasm in the nucleus and cytoplasm outside the nucleus and bounded by the cell wall or plasma membrane.

1.2.

Biodiversity

1.2.1.

Eukaryotes

It has been found that the cell theory is still limited to only one group of animals which body is composed of only a single cell (Protozoa) or the aggregations of cells, the tissues (multicellular, Parazoa, Metazoa) and such animals are designated as the "Eukaryotes". They include the animals from Protozoa to Chordata. Each cell of their body consists of a definite nucleus with a well defined nuclear membrane, cytoplasm with organelles and inclusions and a cell wall or plasma membrane. The light and electron microscopy has revealed that the eukaryotic cell contains three basic structures, outer plasma membrane, central nucleus and peripheral cytoplasm (Fig. 1.1). Lysosomes

SmoothER

f~---"''''';''':''~;p.

Goigi body

Mitocondria Ribosomes Nudeolus GranularE

Nudear

Chromatin

.... : envelope Plasma membrane

Fig 1.1 Eukaryotic animal cell.

The plasma membrane is composed of paired outer protein and inner lipid layers . The proteins include integral proteins (distributed among lipid molecules), transmembrane proteins (extend from plasma membrane to cytoplasm), peripheral proteins (associateted with polar heads of lipids) and glycoproteins (protruded in extracellular fluid forming glycocalyx). It is characterized with permeability and transport activity bearing receptor system and immune particles (Fig. 1.2). The nucleus is a large organelle that contains chromatin

3

Introduction

Outer protein layer

embedded in the nucleoplasm and externally bounded by' a nuclear membrane containing pores all over a surface. It is a central and commanding, in fact, a heart of a cell performing various metabolic, developmental and hereditary activities.

The chromatin is an aggregation of chromosomes. The number of chromosome is constant for each species. Male and female gametic nuclei have Fig1.2 Plasma membrane: (A) Unit membrane model and haploid while somatic cells have diploid (8) Greater membrane model sets of chromosomes. Haploid set of chromosomes of a gamete forms a genome of a species. A particular species is characterized with a fixed number of chromosomes, for example, mosquito, Culex pipiens - 6, housefly, Musca domestica -12, hydra, Hydra vulgaris - 32, frog, Rana esculenta - 26, rabbit, Oryctolagus cuniculus - 44, and man, Homo sapiens - 46. In human female 44 (22 pairs) autosomes (nonsex) and 2 (one pair) alike sex chromosomes, XX while in man equal number of (44) autosomes but one long X and other short Y chromosomes are present. All chromosome pairs of a particular species represent a unique morphological picture, called karyotype. There are giant chromosomes, polytene in salivary gland cells of dipteran flies and lampbrush in oocytes of some animals(Fig.I.3).

/""

Chromatids

(A)

(8)

(C)

Fig. 1.3 Animal chromosome: (A) Metaphase chromosome, (8) Polytene chromosome and (C) Lampbrush chromosome

During metaphase each chromosome consists of two symmetrical long rod - like structures, called the chromatids. They are attached together by a centromere. According to the solenoid model of chromatin, each chromatid contains a single DNA molecule which is tightly bound to an equal mass of globular histone protein molecules forming together the nucleosomes. The nucleosomes are the fundamental packing unit particles (beads) of the chromatin. Each nucleosome is a disc-shaped beaded structure of about 11 nm diameter and 5.7 nm in length consisting of 2 copies of each 4 nucleosome histones - H 2A, ~B, H3 and H 4 . This histone octamer forms a protein core around which

Techniques in Life Sciences

4

the double stranded DNA helix is wound 13/4 times containing 146 base pairs in human chromosome. Enormously coiled helical-like molecule of the deoxyribose nucleic acid (DNA) is so tightly wrapped around a skeleton of histone that besides its enormous length, it occupies a small space within a chromosome. The DNA molecule is capable of producing a new alike DNA molecule as its exact true copy and a molecule of ribonucleic acid (RNA) as its complementary copy through the process of replication (Fig. 1.4).

2 Turns of DNA per nucleo-

-

(A)

(8)

(C)

Fig. 1.4 Structure of a DNA : (A) Nucleosome, (8) DNA helix and (C) DNA molecular structure

The DNA molecules, according to a "DNA structural model" proposed by Watson and Crick (1953), are the double stranded helical like polymeric structures. Each molecule is composed offour types of deoxyribonucleotides - the deoxyadenylic acid, deoxyguanylic add, deoxycytidylic acid and deoxythymidylic acid. The DNA molecule (polynucleotide chain) is a double stranded helical - like structure of 20A diameter, making a complete tum every 34 Aalong its length with a 3.4 A intemucleotide distance and therefore, consisting a series of ten nucleotides per tum. The nucleotides are joined in a chain by phosphodiester bridges or bonds. The deoxyribosephosphate chains are lying outside forming a ribbon like backbone and purine and pyrimidine bases on inside of the helix between both the backbones as the transverse bars. Both tne polynucleotides within a DNA molecule are held together by the hydrogen bonds between specific pairs of the purine and pyrimidine bases which strictly allow pairing of adenine with thymine by two hydrogen bonds and between guanine and cytosine by three hydrogen bonds. The RNA (Fig. 1.5) is a polynucleotide molecule similar in chemical composition to that of DNA except that the pyrimidine base, thymine is replaced by uracil and sugar, deoxyribose is replaced by the ribose. The RNA molecule is, therefore, composed of the two purine and two pyridine bases- adenine (A), guanine (G), and cytosine (C) and uracil (U) respectively. They combine individually with a ribose sugar molecule and form the ribonucleosides such as adenosine, guanosine, .cytidine and uridine. Again each nucleoside links with a molecule of phosphoric acid independently and form the ribonucleotides l~ke adenylic acid, guanylic acid, cytidylic acid and uridylic acid respectively. The RNA molecules are replicated from the DNA molecules bearing complementary bases through the process of transcription. Except genomic RNA of some viruses, they have no ability of self-replication like the DNA. The RNA molecules

5

Introduction 3'

A s·

occur in three forms, ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA).

The nucleus is, therfore, a seat of genes forming a genome of an animal. It bears a unique gene dictionary with well established genetic code and solely functions as the regulatory and controlling centre of the protein synthesis .. through which it determines and maintains hereditary (species/ ~ilccj(ron (A) (8) family specific) characters from one Fig 1.5 RNA molecular structure: (A) rRNA and generation to another, quite (8) tRNA clover-leaf model meticulously. The nucleoplasm is rich in the nuclear enzymes involving in DNA replication and ribonucleic acid (RNA) synthesis processes.

The cytoplasm consists of all the cellular contents between the plasma membrane and the nucleus. It has two components, the cytosol and organelles. Cytosol is the fluid of cytoplasm containing water, dissolved solutes and suspended particles of both organic and inorganic nature. The organelles are embedded in the cytosol consisting of cytoskeleton, ribosomes, endoplasmic reticulum, Golgi complex, lysosomes, peroxisomes and mitochondria, specialized to conduct specific functions. The cytosol is the site of many chemical reactions required for cell's existence. The cytoskeleton is network of fine protein filaments (microfilaments, microvilli, intermediate filaments, microtubules, centrosomes etc) extended throughout the cytosol. The cytoskeleton provides a structural framework for the cell, serving as a scaffold and determines a shape of the cell. It also facilitates movement of the organelles within the cell. The microtubules form cilia and flagella externally as the vehicals of transport from one place to other. The ribosomes are the site of protein synthesis containing more than 50 proteins and some, in addition; mRNA. They are formed in the nucleolus. They may attach to the endoplasmic reticulum (ER) forming granular endoplasmic reticulum. The endoplasmic reticulum (ER) forms a fine network of folded membranes extending from nuclear membrane to throughout the cytoplasm. The ER are flattened or tubular structures and occur in two forms smooth or rough (granular endoplasmic reticulum).

The Golgi complex consists of 3 to 20 Golgi cisternae or membranous sacs or tubules like structures. They form a protein synthesizing system along with ER and ribosomes. The lysosomes are the membranous vesicles that are formed from the Golgi complex containing more than 60 kinds of powerful digestive and hydrolytic enzymes that can defend with a large variety of endogenous or exogenous molecules, and microorganisms. The lysosomal

6

Techniques in Life Sciences

membrane is equipped with active pumps that move H+ into lysosome. Its internal pH is about 5 which is about 100 times more acidic than the cytosol (pH of 7).

The peroxisomes are similar to lysosomes but smaller in size and contain several oxidases capable of removal of hydrogen atom from various organic substances. The proteasomes are the small membranous bodies filled with (unneeded, faulty, damaged) protein molecules and some proteases. The mitochondria are the power houses of the cell generating most of the energy in the form of ATP. They consist of outer mitochondrial membrane and inner mitochondrial membrane with small fluid filled space between them. The inner membrane contains a series of folds called, criste. The central space is filled with matrix. Inner membrane is the site of cellular respiration.

1.2.2.

Prokaryotes

The organisms of another group are although unicellular, their cell body organization differs from that of the eukaryotic cells. The cell body of this group of organisms consists of a single circular double stranded DNA molecule forming a chromosome embedded in the clear opaque region called the nucleoid, encircled by the dense cytoplasmic mass simply and never by the definite nuclear membrane. The essential components of the typical eukaryotic cells such as, the nuclear envelope, nucleoplasm, nucleosomes, centrioles, nucleoli, membranous cytoplasmic organelles (ER, GB, mitochondria), and internal cytoskeleton -like structures, are totally lacking. One more striking difference is that the eukaryotic cells multiply through mitosis and meiosis types of cell divisions but the prokaryotic cells always mUltiply amitotically. These animals are, although single celled animals like the protozoans, grouped independently as the "Prokaryotes". They include mycoplasma (PPLO), bacteria and cynobacteria or blue-green algae. They, thus represent altogether different cellular organization which is too much primitive than that of the eukaryotic cells. protein molecules

Ribosomes

Protein molecules Outer membrane

'~::::~~~II:l1I1I1[] Innermem~ne Cell wall

plasma membrane chromosome Fig 1.6 Prokaryote Cell: (A) Escherichia coli and (8) Gram-negative bacterial cell wall

The prokaryotic (bacterial) cell structure is revealed by the electron microscope. The wall or outer covering is composed of three layers, i) the plasma membrane with some infoldings forming mesosomes and chromatophores in side the cytoplasm; ii) the cell wall is laid on the plasma membrane as a thick and composed of proteins, lipids and polysaccharides like chitin. It is

7

Introduction

differentiated into two sub layers, a) an inner sub-layer of gel, proteoglycan or peptidoglycan above a plasma membrane after leaving a periplasmic space and b) the outer sub-layer composed of lipid bilayer traversed by 'porin' polypeptide molecules; and iii) the capsule is the outer most layer lying above a cell wall in some bacteria. It is a thick, gummy, mucilaginous gel or slime layer secreted by a plasma membrane (Fig. 1.6). Internally, the cell body is filled with the cytoplasm or cytosol containing water, prteins, enzymes, lipids, carbohydrates, mRNA, tRNA and rRNA, RNA polymerase molecules, ribosomes and in some bacteria, plasmids also. Some bacteria like Escherichia coli are motile and contain one or more flagella of about 15 to 20 nm in diameter and 201m in length and serve as the swimming organs. Some gram-negative bacilli contain very fine appendages called, the fibriae or pili as the adhesive structures. Some gram positive bacteria however, contain tubular, pericellular and rigid appendages, the spinae formed of 'spinin' protein moiety. som~

1.2.3.

Viruses

There is, in addition, a third group of infectious, sub-cellular and ultramicroscopic particle-like organisms, the "viruses" which do not fit in either eukaryotes or prokaryotes as they do not possess typical cell wall, cytoplasm, cytoplasmic organelles and nucleus, though, they possess a genetic material in the form of nucleic acid(s). The viruses are fully capable of reproduction, protein synthesis and for their multiplication, they always require the host cell and maintain the obligatory parasite - host relationship with the eukaryotes and prokaryotes. The viruses are so minute (ranging from 300 - 3000

Ain size) that they can only be

Surface envelope protein Transmembrane envelope protein

Capsid

Lipid membrane bilayer

I

(8) (A) ..1-_ _ Head

Neck and collar Nudeic :-H'---.f-f-+{ add

(D)

End plate (C) End fibres

Fig.1.7 Viruses :(A) Retrovirus, (8) Tobacco mosaic viruses, (C)Enveloped virus (D) T4 bactetriophage

8

Techniques in Life Sciences

.,, -, ..

ONAVIRUSES

'. '.'

.~- ,~,

I

' . ....

'," •• ' .i, \I

'~'.' t" \ ,,, '. • • •.,., ';'1

. (A)

-

,". .... , (8)

(C)

(0)

RNA VIRUSES

(E)

(I)

Fig. 1.8 Viruses:

(F)

(J)

(H)

(G)

(K)

(L)

(A) Adenovirus, (8) Papovavirus, (C) Poxvirus, (0) Herpevlrus, (E) Rhabdovirus, (F) Retrovirus, (G) Togavirus, (H) Orthomyxovirus (I) Reovirus (J) Paramyxovlrus, (K) Arenavirus and (L)Picomavirus

observed under electron microscope or by X-ray crystallography. The infectious virus particle is called a viron and it consists of a core of only one type of a nucleic acid, either DNA or RNA enclosed in a protein capsule, called capsid. The capsid is made up of several capsomeres, each representing one or more polypeptide molecules as the structural units. The capsomeres may be of various shapes such as - prism-like triangular, hexagonal, pentagonal, oval, lobular or globular. Due to which the viruses retain a) icosahedral (20 sided in capsid with penta and hexamerecapsomeres as found in Bacteriophage 0, poliovirus, adenovirus); b) helical or cylindrical (rod shaped capsids, tobacco mosaic virus, helical shaped influenza virus) and complex symmetrical forms (with indistinct caps ids pox viruses and tadpole shaped viruses - T even phages of E. coli, bullet shaped rabbis viruses). The tadpole virus is represented by an icosahedral head, helical tail sheath, hexagonal end plate and rod-shaped tail fibres (Fig. 1.7, 1.8). The viruses that parasitize the bacteria are called bacteriophages or phages. The life cycle of the bacteriophages passes through the lytic virulent cycle or lysogenic phage. The lytic cycle occurs after viral infection causing bursting and death of a host bacterial cell and release of

Introduction

9

the new infective virulent phages such as - the T4 and other T even phages from E. coli. The lysogenic cycle does not cause lysis of a host cell after infection eg., Lambda phages, PI phages.

2

TISSUE CONCEPT

The histological studies reveal that the cells of various organs of a body are basically similar in structure and functions. They uniformly consist of an outer cell wall or plasma membrane, centrally placed nuclei and some organelles like, mitochondria, Golgi bodies, endoplasmic reticulum, ribosomes, and Iysosomes embedded into the cytoplasm. These studies have also demonstrated that they uniformly perform various mechanisms like, protein synthesis, metabolic activities, energy transformation, storage of organic and inorganic substances, duplication and equal division of nuclei, nucleoplasm, chromosomes and nucleic acids during meotic or mitotic cell division. At the same time, thorough histological studies explore that the cells exhibit some specializations in their structure and functions. The organization of cells in different p~bphils, , plas~a cells, '·:Hs~~~s~:get~;~i(:mti~~lesl:~es~eis. ~~ .;: &intangled c¢iis) '. ~elan9Cytes;·' fuast ceih .. nerve~:. ~"·' ..... ,0

. . '

•

.

•

)'.
. A y h i n C -+ Proline

Urea

Carbamoyl phosphate synthetase

Urea NH~

I

yH2 NH z

C=NH + yH 2-NH 2

9H2Hz 9 H-9-NH~

yH2 Hz 9 H-9- NH2

COOH Arginine

o 0

II II H2N-C-0-f-0 Carbamoyl phosphate

0-

COOH I H9 CH

COOH Arginine

Ornithine transcargamoylase

I

Argininosuccinase NH I

COOH Fumaric acid

COOH I

y-NH-yH yH 2NH t CH t I

(3)

Argininosuccinic acid synthetase

CH t I

H-9- NH2 COOH

AMP+ pP. COOH

HaN-

t-

H I CH t

tOOH Alp.rtie acid

Fig 13.1 Urea biosynthesis through ornithine cycle

yH2 COOH

Argininosuccinic acid

285

Detection of Excretory Substances

(C) Urecotelic Animals - Terrestrial and desert animals, reptiles and birds excrete uric acid (or urates) as the major nitrogenous waste. Insects and snails also excrete solid pellets of uric acid. Snails like Limnaea and Paludina excrete uric acid after returning from terrestrial to aquatic habitat. It is non-toxic and can be stored in the body for a long time. It can be removed in a solid form without any loss of water from the body. It is in fact, a physiological adaptation of those animals living in warm atmosphere (desert) and water conservation is vital need of these animals. Their skin is also impermeable and prevents water loss from the body. In terrestrial insects water is obtained through a food in a little quantity and it is reabsorbed by Malpighian tubules and rectal pads efficiently. The uric acid is derived from a metabolism of purines (adenine and guanine) and/or from the proteins by utilizing glycimine, glutamine and aspartate as the substrates. The reaction proceeds as follows: Adenine-+ Hypoxanthine -+ Xanthine -+ Guanine -~ Uric Acid. Sometimes, degradation of uric acid may occur in the body as follows: Uricase

Allantoinase

~

~

Allantoicase Urease

~

~

Uric acid -+ Allantoine -+ Allantoic acid -+ Urea -+ Ammonia + C02. Other nitrogenous compounds include - amino acids, allantoine, allantoic acid, guanine, adenine and they excreted through urine with the major nitrogenous products. After maintaining a hydromineral (water -electrolyte) balance (osmoregulation) of the body precisely, excess mineral ions sodium, potassium, calcium, magnesium, chloride, sulphates, phosphates etc are also eliminated with excess water in the form of urine.

Phosphocreatine

Creatine

CH'-N-CH':~

L

/C=O

HN= Nfl Creatinine Fig 13.2 Formation of creatine and creatinine

H P0 S

4

286

Techniques in Life Sciences Ionic Composition of Human Normal Urine (meq.lL) Constituents Sodium Potassium Calcium

2

Amount 143 35 6

Constituents Magnesium Chloride Sulphate

Amount 09 136 20-60

QUALITATIVE DETECTION Collection of Urine Sample Midstream urine of about 20 ml volume should be collected into a clean, well washed and dried (sterilized) bottle or any glass container with a stopper to avoid bacterial contamination. Bacteria may convert urea into ammonium carbonate and become unsuitable for determination of urea, ammonia, pH, and nitrogen. Bacteria and yeasts may utilize glucose, phosphates may be precipitated and urobilinogen may oxidize to urobilin. Add 10 ml of concentrated hydrochloric acid to a 24 hour urine specimen for preservation. Now the sample is suitable for the determination of urea, ammonia, total nitrogen and calcium. It is always advisable to keep urine specimen at 4°C in the laboratory. Women should not collect urine during their menstrual period. Catheterization may allow microorganisms into the bladder so better to avoid sample collection during such condition.

2.1

NORMAL CONSTITUENTS

2.1.1. Urease Test for Urea Principle Urease, the urealytic enzyme carries out break down of urea into ammonium carbonate. It causes increase in alkalinity so that the pink colour in solution reappears distinctly.

Procedure D D D D D

Take 5 ml urine or unknown solution to be tested. Add 2 ml of 2% Na 2C03 solution. Add 4 drops of phenolpthalin solution as a indicator. Add drop by drop (5 - 10 drops) of 1% acetic acid. Add a pinch (20 mg) of urease powder (commercially available).

Observations and Inference Phenolpthalin gives a light pink colour to solution while action of acetic acid decolorizes the solution which reappears as a dark pink colour again after a reaction of urease.

2.1.2 Oxidation or Murexide Test for Uric Acid Principle Uric acid is oxidized to dialuric acid and alloxan. Alloxan undergoes condensation and forms, alloxanthin. Alloxanthin reacts with ammonium or potassium hydroxide and forms, a purple coloured compound, murexide.

Procedure D D D

Take 1 ml of urine or a given solution in a clean, dried test tube. Add 2-3 drops of concentrated nitric acid. Evoporate the solution on a low flame of spirit lamp till a complete dryness.

Detection of Excretory Substances

287

D Cool and add a drop of dilute potassium/ammonium hydroxide solution separately. D Warm the solution for 2 - 5 min. Observations and Inference After step 3, red or yellow coloured residue is formed. After treatment of NH4 0H or KOH. colour changes to purplish red or purplish violet indicating presence of uric acid. Note - Uric acid is an excretory end product in insects. To demonstrate it, dissect out Malpighian tubules with hind gut in a petri dish, put few drops of KOH solution and crush to form a paste. Take a filter paper, fold it and take hind gut- Malpighian tubule extract on a filter paper and put few drops of AgN0 3 solution. Due to reduction a black coloured precipitate is formed indicating presence of uric acid as an excretory end product in insects. 2.1.3 Urease Test for Ammonia Principle The enzyme urease converts urea into ammonia. Procedure D Take 2-3 ml of given solution in two separate test tubes. D Add phenolpthaline indicator solution to each test tube. D Take one of the two test tubes and add a pinch of urease powder. Observations and Inference Due to phenolpthaline, solution turns milky in each test tube showing presence of urea. After urase reaction the above solution turns red indicating formation of ammonia. 2.1.4. Jaffe's Test for Creatine Principle Creatine is converted into creatine picrate and red colour appears in alkaline medium. Procedure D Take 5 ml of given solution or urine in a test tube. D Add 1 ml of saturated picric acid. D Add 1 ml of sodium hydroxide solution. Observations and Inference Finally solution turns red indicating presence of creatine. 2.2

ABNORMAL CONSTITUENTS

2.2.1 Benedict's Test for Glucose Principle Due to Benedict's reaction glucose reduces cupric ions present in the reagent to cuprous ions and as a result, original blue colour of the reagent changes to green, yellow, orange or red in relation to a concentration of glucose present in the urine. Procedure D Pipette 5.0 ml of Benedict's solution in a clean test tube. D Add 0.5 ml or 8 drops of fresh urine with the help of Pasteur pipette. D Heat the tube on spirit lamp/burner flame or keep in boiling water bath till the mixture boils, for 5-10 min. D Cool under tap water or by placing in a beaker containing tap water.

288

Techniques in Life Sciences

Observations and Inference In diabetic condition, the glucose is present in urine. Formation of green-light yellow precipitate indicates presence of traces of sugar (glucose) about 250 -- 500 mg/dl. Formation of green- dark yellow precipitate indicates presence of sugar (glucose) about 500 - 1000 mg/dl. Formation of yellow-orange precipitate indicates presence of sugar (glucose) about 1000 - 1500 mg/dl. Formation of orange -red precipitate indicates presence of sugar (glucose) about more than 1500 mg/dl. 2.2.2 Osazone Test for Lactose Principle Urine lactose reacts with phenyl hydrazine hydrochloride in acidic medium and at high temperature produces lactosazone crystals. Procedure o Take 5 ml of urine in a clean test tube. o Add a few drops of glacial acetic acid, use litmus paper to confirm acidic nature of urine. o Add I g of sodium acetate and phenyl hydrazine hydrochloride (2: 1) mixture. o Keep in boiling water bath for 30 min. o Transfer a tube in a beaker containing tap water for cooling. Observations and Inference Formation of fibrous crystalline precipitate at a bottom of a test tube indicates presence of lactose. Fibrous crystals can be examined under microscope also.The positive reaction appears in persons with deficiency of lactase enzyme. Positive reaction also occurs in the lactating women and 3 to 5 days old infants. 2.2.3 Seliwanoff's Test for Fructose Principle HCL reacts with fructose forming a derivative of furfuraldehyde. The latter reacts with resorcinol and forms a red coloured compound. Procedure o Pipette 5 ml of Seliwanoff's reagent in a test tube. o Add 0.5 ml of urine. o Place in boiling water bath for 5 min. Observations and Inference Appearance of red color indicates presence of fructose in urine. It appears in persons with hepatic disorders. 2.2.4 Sulphosalicylic acid Reaction for Protein Principle Urine proteins react with a sulphosalicylic acid and form a precipitate. Procedure o Take 3 ml of clear urine in a test tube. o Add 2-3 drops of 3% sulphosalicylic acid.

Detection of Excretory Substances

289

o

Add 2-3 drops of glacial acetic acid (to digest phosphorus, if present). Observations and Inference Formation of precipitate after treatment of both, sulphosalicylic acid and glacial acid confirms presence of protein. About 150 mg/24 h is excreted mostly low moleculr proteins like albumin and gamma-globulin. Glamerular damage causes increase in urine protein concentration, proteinurea.

2.2.5 Rothera's Test for Ketones Principle Nitropruside used in the test reacts with both, acetone and acetoacetic acid in the presence of alkali, ammonium hydroxide, to produce a purple coloured compound. Rothera's Mixture Sodium nitroprusside 0.75 g 20g Ammonium sulphate Mix both powders and pulverize and store at room temperature. Procedure o Take 5 ml of urine in a clean test tube with the help of Pasteur pipette. o Add I g of Rothera's mixture (powder) and mix well. o Add 1-2 ml of ammonium hydroxide (liquor ammonia solution) slowly along wall of a test tube. Observations and Inference In diabetic condition, a pink purple ring at the interface appears distinctly and after shaking the tube, colour spreads through out the solution suggesting presence of ketone bodies or ketonuria. 2.2.6 Hay's Sulphur flower Test for Bile Salts Principle Sulphur particles sink on the bottom of a test tube containing urine indicates presence of bile salt. In normal condition sulphur particles float on a surface of the urine. Procedure o Take 10 ml of urine in clean and dry test tube (15 X 125 mm). o Sprinkle a little dry sulphur powder on to surface of the urine. Observations and Inference Shinking of sulphur particles on bottom of a tube indicates presence of bile salts in urine under jaundice due to enormous increase of bilirubin in the blood. Bile salts are distinctly present during hepatic and post-hepatic conditions. 2.2.7 Harrison Spot Test for Bile Pigments and Urobilinogen Principle On addition of barium chloride solution to urine, It combines with sulphate radicals in urine and precipitate of barium sulphate is formed. Ferric chloride oxidizes yellow bilirubin to green biliverdin in the presence of trichloro-acetic acid.

Techniques in Life Sciences

290 Procedure

o o o o o

o

Take 4 ml of urine in a centrifuge tube (A) by Pasteur pipette. Add equal amount of I Og/dl barium chloride solution, mix well. Centrifuge at 1500 rpm for 10 min. Remove supernatant in another tube (B) for a test of urobilinogen. Add 2 drops of Foucht's reagent (commercially available) on a precipitate taken from tube A on to a filter paper. Add about 0.5 ml of Ehrlich's reagent (commercially available) to the supernatant.

Observations and Inference Precipitate on filter paper (step 5) colour changes to green showing presence of bile pigments. Appearance of pale pink to cherry red colour indicates presence of low or high concentration of urobilinogen respectively.

2.2.8 Peroxidase Test for Occult Blood in Urine Principle Haemoglobin and myoglobin catalyse (like peroxidase) the oxidation of an indicator. The colour changes from orange-yellow to green to dark blue.

Procedure

o o o o

Take 3-5 ml of urine in two clean and dry test tubes. Add in one test tube a few drops of an indicator, 3,3,5 - tetramethylbenzidine and wait for 10 min. Add in another test tube, a few drops of an indicator, tetramethylbenzidine peroxidase. Take a little quantity of dry benzidine powder in a third dry test tube, add 1 ml of glacial acetic acid and add 2 ml of urine sample and a few drops of hydrogen peroxide from a freshly opened bottle.

Observations and Inference

o

Change of colour from orange to red of the solution in the first and second test tubes indicates presence of blood in urine. Solution of third test tube also develops green to dark blue colour and after 5 - 10 min turns to purple/red colour indicating presence of blood in urine.

3.

QUANTITATIVE DETECTION

3.1 Jaffe's Reaction for Serum Creatinine Principle Creatinine in alkaline medium reacts with picrate to produce orange colour. This colour absorbs maximum light at 492 nm. The, rate of increase in absorbance is directly proportional to the concentration of creatinine in specimen. Alkaline medium creatinine + Picrate ~ (Orange coloured compound)

Reagent composition I Sodium picrate 2 Sodium hydroxide 3 Creatinine standard

7.7 mMol1L 500 mMollL 2 mg%

Detection of Excretory Substances

291

4. Mix equal volume of reagent 1 and 2 and prepare as working solution half an hour before the analysis. Store the stock reagents at room temperature. Procedure Sample o Pipette into test tubes Standard Reagent 4 1.0 m!. 1.0 m!. Standard 50.0 III Sample 50.0 III o Mix the reagents and start stopwatch immediately. Record the absorbance of assay exactly at 30 seconds after standard /specimen addition and then again at 90 seconds. Take the readings at 492 nm. Calculations The change in absorbance per minute (Abs) of standard and specimen (S) was calculated as follows: Abs =Abs. at 90 Secs. - Abs. at 30 Secs. Serum creatinine =

Abs. of unknown x 2 mg% Abs. of Standard

Clinical significance Creatinine is a waste product formed in muscles and excreted through kidneys in urine. Consequently, the blood levels of creatinine depend on kidney function. Elevated levels are observed when kidney function is impaired. 3.2 DMA Uric Acid Uricase Technique for Serum Uric Acid Principle The DMA uric acid uricase procedure is based on the oxidation of uric acid by uricase to allantoin, carbon dioxide, and hydrogen peroxide (~OJ Hydrogen peroxide in the presence of peroxidase causes the oxidative coupling of 4-aminoantipyrine (4-AAP) and 2-hydroxy3, 5-dichlorobenzene sui phonic acid (HDBS) to form a red chromogen, which is proportional to the amount of uric acid present. Reagents 1. 2-hydroxy-3, 5-dichlorobenzene sulfonic acid 3.0 m mollL 2. 4-aminoantipyrene 0.4 m mollL 3. Uricase (Yeast) 160UIL 4. Peroxidase (horseradish) 500UIL 5. Sodium azide buffer surfactant preservative Note: Sodium azide may react with lead joints in copper drain lines to form compounds. Drains should be well flushed with water when discarding the reagent. 6 Add the amount of deionized water to each vial as stated on the label swirl to dissolve the contents. Allow to stand 10 minutes at room temperature before use. Procedure o Pipette 2.0 ml of uric acid reagent into labeled test tubes for each sample. o Add 0.05 ml of standard, control and sample separately to the respective test tubes, mix gently and incubate the test tubes at room temperature for 10 minutes.

292

o

Techniques in Life Sciences Take the readings at 520 nm by setting zero with de-ionized water as a blank.

Calculations To calculate the uric acid concentration of serum by using following formula -

Abs. of unknown x Cone. of Std. Uric Acid (mg/dL) =

Abs. of Standard

Sensitivity Based on an instrument resolution of A= 0.001, this DMA uric acid uricase procedure has a sensitivity of 0.03 mg./dL.

Clinical Significance Uric acid is one of the components of the non-protein nitrogen fraction of plasma. It is a waste product derived from purine metabolism. Normally, about half of the total uric acid is eliminated each day by way of urinary excretion and by destruction in the intestinal tract by microorganisms. Increased uric acid levels are most commonly associated with nitrogen retention and increased levels of urea, creatinine and other non-protein nitrogen constituents. These conditions are indicative of poor function. Uric acid determinations are also helpful in the diagnosis of gout. Increased levels are noted whenever there is an increased in nucleoprotein metabolism, such as leukemia and polycythemia or after the intake of food rich in nucleoprotein, such as liver and kidney.

3.3

Glutamate Dehydrogenase (GLDH) Technique for Blood Urea Nitrogen (BUN)

Principle Urease Urea+Hp--~

GLDH NH3 +aKG+NADH ) L-Glutamate + NAD + a KG ( a KG = a-Ketoglutarate; GLDH = Glutamate dehydrogenase)

Reagents Reagent -I 2-0xoglutarate NADH (Yeast) Urease (Jack Bean) GLDH(Beef) ADP Tris Buffer pH 7.9 ± 0.1 at 25°C 100 mM! L Reagent -1/ BUN Standard All the reagents were stored at 2° to 8° C.

7.5mM!L 0.32mM!L 8.000 lUlL 1.000 lUlL 1.2 mM/L

23.4mg/dl

Detection of Excretory Substances

293

Procedure Standard Sample Pipette into test tubes Reagent-I ~ 100 III 100 III Standard 20 III Sample ~ 20 III Incubate at 37°C and read at 340 nm. against distilled water as a blank. Calculation M=AI-A2

Aof Sample

Urea (mg/dL) = -----=--xConc. of Std. (mg/dL)

A of Standard

Clinical significance Blood urea nitrogen is the major end product of protein nitrogen metabolism in animals and humans. It constitutes the largest fraction of the non-protein nitrogen component of the blood. BUN is produced in liver and excreted through the kidneys in urine. Consequently, the circulating levels of blood urea nitrogen depend upon protein intake, protein,catabolism and kidney function. Elevated levels of BUN concentrations are observed in impaired kidney function, liver diseases, congestive cardiac failure, diabetes. infection and diseases which impairs kidney functions. 3.4

Folin Method for Amino Acid Nitrogen in Blood

Principle When the amino acids react with a-naphthoquinone-4-sulphonate in alkaline solution, a brown coloured substance is produced and its absorbance can be read at 470-480 nm. Reagents I. Sodium a-naphthoquinone-4-sulphonate: 0.5%, aqueous. Freshly prepared. 2. Borax, sodium tetraborate 2% aqueous solution. 3. Sodium hydroxide, O.IN, aqueous 4. Phenolphthalein solution in ethanol, 0.25%. 5. Acid formal dehyde solution: 0.3N HCl containing 3 ml of 40% formaldehyde per litre. 6. Sodium thiosulphate, 0.05N 7. Stock amino acid solution: It contains 0.2 mg amino acid nitrogen per ml in 0.2 per cent sodium benzoate dissolved in 0.7N HC!. 8. Equimolar mixture of glycine and glutamic acid (glycine - 53.6 mg and glutamic acid - 105 mg in 0.2% sodium benzoate in 0.7 N HCI, making up to 100 ml with the same solution) is recommended to obtain bright colour of the test. 9. Standard: Dilute 3 ml of stock solution to 100 ml with 0.2% benzoate in acid. One ml of this solution contains 6 fJg of amino acid nitrogen. Procedure o Pipette 5 ml of folin tungstate acid filtrate prepared from a plasma into a test tube o Add a drop of phenolphthalein and then 0.1 N sodium hydroxide, drop by drop, until a permanent pink colour is produced. o Treat 5 ml of the amino acid standard (containing 0.030 mg amino acid nitrogen) and set

Techniques in Life Sciences

294

up a blank using water instead of blood plasma to check that the sodium tungstate and the acid are ammonia-free. D Add 1 ml of distilled water to get the same volume in all the tubes. D Add I ml of the borax solution to each tube. D Mix and add 1 ml of freshly prepared naphthoquinone solution. D Mix and place the tubes in a bath of boiling water for 10 min. D Remove tubes from bath and keep in cold water for 5 min. D Dilute almost to 13 ml with distilled water. D Add 1 ml of acid formaldehyde reagent, mix and add 1 ml of 0.05 N thiosulphate (adjust pH 9.2 to 9.4 for reaction to develop colour fully). o Make up to 15 ml with distilled water. Mix and keep for 10- 30 min at room temperature till the brown colour is developed. o Read absorbance of colour at 470 - 480 nm using a blue filter. Calculation mg amino acid nitrogen/IOO ml of blood plasma

=

=

Reading of Unknown Reading of Standard

100 X 0.030 X - 0.5

Reading of Unknown

X6. Reading of Standard A suitable standard curve can be prepared by using a solution containing 20~g amino acid nitrogen per ml by diluting 10 ml of stock standard solution to 100 ml.as follows:

mg amino acid nitrogen/IOO ml ml standard (I ml ml Distilled water

= 20 ~g)

o

2

4

6

8

10

12

o

0.5

1.0

1.5

2.0

2.5

3.0

5

4.5

4.0

3.5

3.0

2.5

2.0

Clinical Significance The normal range of plasma amino acid nitrogen in man is about, 3.4 to 7.8 mg/I 00 ml. An increased level of amino acid nitrogen even up to 15 to 25 mg/IOO ml of plasma is commonly found in acute hepatic necrosis. Higher concentration of amino acid nitrogen is also recorded in acute infective and toxic hepatitis and advanced cirrhosis. 3.5. Flame Photometrical Estimation of Sodium (Na) and Potassium (K) Principle At a higher temperature atoms of various elements dissociate from their salt to become higher energy state and emit specific spectral bands, which absorb through proper interference filters or spectral barriers in a photo detector. The absorbance is proportional to the elements concentration. The emitted light of sodium absorbs maximum at 589 nm potassium at 404.5 and 766.5 nm and calcium at 620, 554 and 422.7 nm. The presence of various other elements as contaminants makes it difficult to trace out each individual

295

Detection of Excretory Substances

analyze. However, by suitable multiplication of photo detector satisfactory results comparable to Ion Sensing Electrometry (ISE) are obtained A) Determination of Serum Na/K

Reagents Standards are diluted 1 to 100. Therefore the values are comparable only when test sample is similarly diluted 1 to 100 with distilled water.

Composition 500ml First 1 Sodium 20 mMolIL 1 Potassium 2mMolIL Second 500ml 2. Sodium 140mMoIIL 2. Potassium 4mMoVI Third 500ml 3. Sodium 160mMolII 3. Potassium 6 mMoVI All standards are diluted I to 100 in 1 mMol1L lithium nitrate, containing preservatives and surfactants. Samples: The most satisfactory dilution to be used for sodium may be 1 in 100, 1 in 200 even 1 in 500; for potassium it is usually lower, often 1 in 50. Standard solutions: Stock sodium solution: 1,000 milliequivalents per litre. 58.5 g of sodium chloride per litre of solution. Stock potassium solution: 100 milliequivalents per litre, 7.46 g of potassium chloride per litre of solution. A series of stock standards can be prepared from these as follows: mEQ. Na / litre 120 130 140 150 160

ml stock A 60 65 70 75 80

ml stock B 25 25 25 25 25

meQ. Kllitre 3 4 5 6 8

ml stockB 15 20 25 30 40

ml stock A 70 70 70 70 ·70

Make up to 500 ml with distilled water. When carrying out series of determinations, insert an appropriate standard solution at frequent intervals to ensure that constancy of reading is being obtained.

Functioning of Flame Photometer. Sample is picked up from a beaker by a compressed stream of air as the control valve is pressed and enters an inlet tube and sprays at the spray chamber. Large droplets flow to waste through a drainage tube. Gas is introduced in the spray chamber through the inlet tube which is connected to the gas pressure stabilizer and to a control valve. The gas-air mixture bums in a flat flame and the hot gases pass up to a well ventilated chimney. Gause

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in the burner tube prevents serious firing back of the flame. The light emitted by a flame is collected by a reflector and focused by a lens through the interchangeable optical filters on to an EEL barrier layer selenium photocell. This is connected in a series with a calibrated potentiometer and a galvanometer unit. A glass window is placed between the lense and the filter for cooling purposes.

Procedure

o

Pipette in the tubes labeled as follows:

Reagents (ml)

o o o

o o o o o o

o o

Test

Std. 1

Std.2

Std.3

Distilled water 10 10 10 10 o. I Serum/plasma Std. 1 - 120/2.0 0.1 0.1 Std. 2 - 14014 .0 0.1 Std. 3 - 160/6.0 Mix and transfer to beakers for flame photometric determination. Put on a main switch of the flame photometer. Put on air compressor and adjust the required air pressure, by adjusting the knob meant for air. Introduce distilled water, through automiser. Put on gas and control the flame by adjusting the knob meant for gas. It is adjusted till the flame is divided into five sharp cones. Adjust proper filters for the simultaneous determination of sodium and potassium. Make zero adjustment by introducingt distilled water. Introduce the standard I - 120/2.0 and by using the knob meant for sodium, the digits 120.0 by using the knob meant for potassium, the digits 2.0 are adjusted. Introduce standard 2 - 140/4.0. If the standards are accurately prepared, the digital display will indicate exact concentration for both sodium and potassium. Introduce standard 3- 160/6.0 and confirm accuracy of standardization. Now introduce the Test and record the readings for sodium and potassium.

B) Determination of Urine Na and K Conduct flamephotometry for determination of urine - Na and K, in the similar way to that of serum Na and K. Use undiluted urine sample instead of serum. Follow the following-

Calculations NaiK mEq/L = Reading X dilution factor. Eg. If urine K diluted 1: 10 then Urine K mEq/L =Reading X 10. Urine Na, mEq/L X 24 h Urine Volume 24 h excretion of urine Na = - - - - - - - - - - - - - - 1000 24 h excretion of Urine K

K, mEq/L X 24 h Urine Volume =Urine ------------1000

Clinical Significance In man, normal range of serum sodium is 133 - 148 mEq/1 and that of serum potassium is

297

Detection of Excretory Substances

3.8 - 5.6 mEq/1. Higher concentration of sodium and potassium is termed as hypematremia and hyperkalemia respectively. Similarly, lower concentration of sodium and potassium is is called as hyponatremia and hypokalemia respectively. Hypematremia occurs during dehydration, diabetes insipidus, salt poisoning, Cushing's syndrome and enlargement of prostate etc. Hyperkalemia is observed in Addison's disease, renal glomerular disease and anuria and oliguria diseases. On the other hand, hyponatremia is found during prolonged diarrhea and vomiting, salt losing nephritis and Addison's disease. Hypokalemia is observed during Cushing's syndrome, renal tubular damage, metabolic alkalosis and malnutrition

3.6 Mercuric Thiocyanate Method for Serum Chloride Principle Chloride reacts with mercuric thiocyanate to give mercuric chloride. The thiocyanate, which is released, reacts with ferric ions present in the reagent to form ferric thiocyanate, which is directly proportional to chloride present in serum/plasma and can be measured at 510 nm. (500-520nm).

Reagents The reagents are for in vitro diagnostic use. Thiocyanate reagent and standard should be stored at temperature indicated on the bottle label. Components and concentrations of working solutions -

Component

Concentration

Mercuric thiocyanate 2.0 mMol. IL Mercuric chloride 0.8 mMol. IL 20.0mMo!' /L Ferric nitrate 45.0 mMo!. IL Nitric acid Stabilizers. surface active agent and Inactive ingredients

Procedure

o

Chloride is stable for 7 days in neatly separated serum/plasma at 2- 8° C. Reaction type End-point Reaction time 1 min. at R.T. Wavelength 510 nm Zero setting with Reagent blank < 0.200 Abs. Blank absorbance limit 0.01 ml. Sample volume 1.0 ml. Reagent volume Standard concentration 100 mMol. /L Linearity 130 mMol.lL

Calculation Chloride in mMollL

=

Absorbance of sample xlOO Absorbance of standard

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298

3.7 Schales and Schales Titration Method for Chloride Principle The protein free filtrate of the specimen is titrated with mercuric nitrate solution in the presence of diphenylcarbazone as the indicator. Free mercuric ions combine with chloride ions to form soluble but non ionized mercuric chloride. After the excess mercuric ions combine with the indicator, diphenylcarbazone to form blue violet coloured complex and colour change of reaction is considered as the end point of titration.

Reagents I. Mercuric nitrate Solution 3.0 g Mercuric nitrate Distilled water SOOml 2N nitric acid 20 ml Make with DW to one litre and store at room temperature in amber-colored bottle. 2. Diphenylcabazone indeicator: Dissolve 100 mg in 100 ml of 95% ethanol. Store at 2°C. 3. Chloride Standard 100 mEq./L.:5.S5 g of analar grade NaCI in 1 litre of dist. Water.

Procedure

o

o o o o o

Pipette in a centrifuge tube 4 ml of distilled water, 0.5 ml of serum, 0.25 ml of 2/3N H2S04 and 0.25 ml of 10% sodium tugstate. Mix well and centrifuge at 3000 rpm for 10 min. Separete the protein free fi Itrate. Pipette in a test tube, 2 ml of protein free filtrate. Add one drop (0.05 ml) of the indicator. Titrate against mercuric nitrate reagent till end point indicated by colourless to violet blue colour and note the titration reading X ml. Dilute standard 1: 10 with distilled water. Pipette 2.0 ml of diluted standard in a test tube and titrate it against mercuric nitrate reagent similar to that for test by using diphenylcabazone indicator and note the titration reading Y ml.

Calculation Serum Chloride, mEq / L

= X ml

X 100

Yml

Clinical Significance Normal chloride range in man is 95 - 106 in serum, 700 to 750 in CSF and 120 - 250 mEq/L in urine excretion for 24 h. Low chloride concentration is observed during vomatings, overhydration and renal diseases while high concentration is noticed during dehydration, renal tubular diseases, congestive heart failure.

3.8. Titrimetric Method for Serum Bicarbonate Principle Serum is added to standard 0.01 N Hel and the loss of strength of the standard acid due to carbonate is determined by titrating the strength of acid against 0.01 N NaOH.

299

Detection of Excretory Substances

Reagents I. Phenol Red indicator - Dissolve 0.1 g of phenol red in 6.0 ml of 0.5 N NaOH and diluted to 100 ml with distilled water.

2. Specimen collection - Collect 2-3 ml blood in a test tube without anticoagulant and allow to clot. Separate a serum and store in a sealed test tube under liquid paraffin.

Procedure

o o o

Pipette 5.0 ml of 1.0 g/dl saline in a conical flask. Add 0.1 ml of the specimen and add 2 drops of the phenol red indicator. Mix well and it is considered as a control. The test is titrated till same colour develops as in the case of control. Pipette 4.0 ml of I % saline in another conical flask. o Add 1.0 ml ofO.OIN HCl mix well. Add 0.1 ml of serum and mix well. o Titrate against O.OIN NaOH until color changes from yellow to red, similar to that in control. Note the titration reading R. ml. Calculation - Serum bicarbonate, mEq/L= (l-R) X 100. Clinical Significance - Normal range in human serum is 21 to 28 mEq/L bicarbonate.

o o

3.9

Daly and Ertingshausen's Molybdate Method for Serum Phosphorous (P) (modified by Wang et al. 1975)

Principle Inorganic phosphorous combines with ammonium molybdate forming a complex of unreduced phosphomolybdate which can be measured at 340 nm and it is directly proportional to the concentration of inorganic phosphorous present.

Reagent composition Reagent A 0.43mMol Ammonium molybdate 213 mMol Sulphuric acid Reagent B Inorganic Phosphorous 5 mg/dl. 1.33 mMoI/L Standard As the reagent contains strong acid, do not pipette by mouth and handle carefully. Protect the reagent from bright light and store at 2 to 8 °C

Procedure o Pipetted into tubes

o

Blank

Standard

Test

Reagent-A 1000 Ill. 1000 III 1000 III 20 III .Distilled water Standard 20 III Sample 20 III Mix well and incubate at 370C for 5 minutes. Take the readings at 340 nm. against reagent blank.

Calculation Phosphorous (mg.dL)

. 0 f standard( mg/dl) = O.D.of test x Concentratzon O.D.of std.

300

Techniques in Life Sciences

Clinical significance In bones more than 80 % of the phosphorous is present in form of phosphates such as phospholipids; nucleic acids and ATP or extra-cellular as inorganic phosphorous. There is generally a reciprocal relationship between serum calcium and inorganic phosphorous levels. The serum phosphorous level increases in renal diseases. hyperparathyroidism and excessive vitamin D -intake, whereas it decreases in rickets, osteomalacia, and hyperthyroidism and in diabetic coma. 3.10 O-Cresolpthalein Complexon Method for Serum Calcium Principle Under acidic condition calcium is chelat~d with cresolpthalein complex, on the addition of alkali results in the formation of a red complex. Magnesium interference is eliminated by addition of 8- hydroxyl-quinoline to the reagent. This method has a pale reagent blank colour compared with other dye binding technique. Reagent composition 1. Stock Cresolpthalein complex Add 20 ml of concentrated hydrochloric acid to the 800 m!. of distilled water. Dissolve 2.5 g of 8-hydroxy quinoline and mix with 42 mg. of cresophthalein and dilute to 1 liter. 2 Diethyl amine solution Prepare freshly every time. Add 0.3 m!. of diethyl amine to 10m!. of distilled water. A. Working calcium reagent Prepare freshly by titrating cresolpthalein complex with diluted diethyl amine to give a mauve colored working reagent. B. Calcium standard - 10 mg/l 00 ml It can be determined by colorimeter, spectrophotometer, atom absorption spectrophotometer or autoanalyser. Procedure o Pipette into tubes Blank Standard Test Reagent-A 1000 ~l. 1000 ~l IOOO~1 Distilled water 20 ~l Standard 20~1

o

~m~

W~

Mix well and incubate at 37°C for 5 minutes. Take the readings at 505 nm. against reagent blank.

Calculation Calcium (mg.dL)

3.11

D.D.of test x Concentratzon . 0 f stand ar d (mg/dl) D.D.of std.

Baron and Bell's Titration Technique for Calcium

Reagents: 1. Alkaline Buffer, pH 12.66.: Mix 20 ml ofJ.1M glycine (1.5 ml g per 200 m!) and 80 ml of 0.1 M sodium hydroxide. 2. Ethylenediaminetetra-acetate: Dissolve 0.98 g of disodium salt in water and make to

Detection of Excretory Substances

301

one litre. 3. Stock Standard Solution (0.1 %): Take 2.50 g of exclusively dry analar calcium carbonate and dissolve in 200 ml of distilled water and add 50 ml ofHCl. Allow to stand overnight and make final volume to one litre. 4. Working Standard (0.01 %): Dilute the 10 ml of stock solution to 100 ml with distilled water. 5. Calcein-thymolphthalein indicator: Grind in mortar 0.2 g of calcein, 0.12 g of thymolphthalein and 20 g of potassium chloride together to a fine powder. Procedure D Pipette 1 ml of serum into 5 ml of buffer solution in a smal1 white dish. Add 1 mg of indicator. D Titrate with the EDTA solution from a micro-burette graduated to 0.0 1 m\. The end point is reached in aqueous solution when the colour changes from yel1ow-green to mauve, with serum, from orange-green to green or pink. Colour is stable for only a few seconds. D Titrate 1 ml of standard and 1 ml of distilled water as a blank in the same way. Calculation Titration of Test -Ttitration of Blank mg calcium/IOO ml serum = X 10. Titration of Standard - Titration of Blank

3.12. Ramsay's Dipyridyl Technique for Serum Iron Principle Ferrous iron gives a pink colour with 2-2' -dipyridy 1. A solution of dipyridyl in acetic acid is added to serum followed by a reducing agent. Proteins are removed by heating in boiling water and then centrifuging or filtering. Ferrous iron gives a pink colour with 22' -dipyridyl.

Reagents I. Dipyridyl Solution: 0.1 % 2-2' -dipyridyl in 3%acetic acid (v/v). 2. Sodium sulphite, 0.1 M: Dissolve 1.26 g of unhydrous sulphite or 2.52 g g of Na~S03. 7H20 in distilled water and make up to 100 ml. Fresh Solution. 3. Standard Solution (100 f.lg iron per mI.): Dissolve 0.498 g offerrous sulphate, FeS0 4 • 7 ~O in distilled water, add 1 ml conc. H 2S04 and make to a litre. Alternately, use a solution of ferrous ammonium sulphate (NH4 )2S0 4 ,FeS04,6HP, containing 0.702 g per litre. 4. Working Standard: Dilute 3 ml of the stock standard to 100 ml with distil1ed water (3f.1g/ml ).

Procedure D D D D D

Mix equal volume of serum, 0.1 M sodium sulphite and dipyridyl reagent in a stoppered tube, useful for centrifugation. Keep in boiling water bath for 5 min. Cool and add 1 ml of chloroform, stopper and shake for a minute. Remove the stopper and centrifuge at 3000 rpm for 5 min. Repeat. Remove the absolutely clear supernatant and read at 520 nm or use green filter.

Techniques in Life Sciences

302 D As blank use water instead of serum. D As standard, use working standard instead of serum. Calculation r.ag Fe per 100 ml of serum Reading of Unknown (Test)

=

X~O

Reading of Standard Note - To obtain a calibrated curve dilute 5 ml of the stock standard to 100 ml with distilled water and set up tubes containing 0.4, 0.8, 1.2, 1.6 and 2 .0 ml of this, make each to 2 ml with water and develop colour as described above and read against the blank. These standard values correspond to 100, 200, 300, 400 and 500 Ilg per 100 m!.

3.13. Bathophenanthrolin Technique for Iron Principle Ferrous iron reacts with bathophenanthroline and forms a complex of a bright pink colour. Thioglycollic acid is used as a reducing agent. Reagents Standard Solution Standard Solution (100 Ilg iron per m!.): Dissol ve 0.498 g of ferrous sulphate, FeS04 •7Hp in distilled water, add I ml conc. H 2S04 and make to a litre. Alternately, use a solution of ferrous ammonium sulphate (NH4 )2S0 4 , FeS04 , 6HP, containing 0.702 g per litre. Working Standard (2Ilg/ml).: Dilute 2 ml of the stock standard to 100 ml with distilled water. Procedure D Pipette 2 ml of serum in a test tube (Test). D Pipette 2 ml of distilled water (Blank). D Pipette 2 ml of working standard (Standard). D Add in all the above tubes, 3 ml of distilled water, one drop of conc. HCI and one drop of thioglycollic acid (80%, aqueous solution). Mix well and stand at room temperature for about 30 min. D Add 1 ml of 30% trichloroacetic acid to each of above tubes, mix well and stand for 10 min. D Centrifuge at 3000 rpm for 15 min. D Take 3 ml of supernatant and add 0.4 ml of 50% sodium acetate and 2 ml of bathophenanthroline (0.02% solution in isopropanol). Mix well and stand for 5 - 10 min at room temperature. D Read at 540 nm the absorbance of colour developed in test, blank and standard. Calculation r.ag Fe per 100 ml blood serum Reading of Test (unknown) - Reading of Blank = X 200. Reading of Standard - Reading of Blank Note - Prepare a standard curve from a 1 to 20 dilution (1 ml = 51lg Fe of the standard solution and put through 0, 0.5, 1.0, 1.5, and 2 .0 m!. made to 2.0 ml with water corresponding to 0, 125,250,375 and 500 Ilg/100ml Fe.

Detection of Excretory Substances

303

Clinical Significance Normal range of Fe is reported about 80 - 17S Ilg/l 00 ml in males and 60 to 160 Ilg/ 100 ml in females while in infants it ranges from ISO to 220 Ilg/100 ml and faIls down to normal range after 3 to 7 years. The blood Fe level shows diurnal rhythm. The levei of blood Fe elevated during haemochromatosis, acute infective hepatitis up to 300 Ilg/ l00ml while low values up to 30 IlgilOOml are reported in iron-deficient anaemia, scurvy, polycythaemia. 3.14. Sodium Diethyldithiocarbamate Technique for Copper Principle The copper is released from its linkage to protein by means of hydrochloric acid, the proteins precipitated by trichloroacetic acid, and the copper extracted from the protein free fluid obtained, into an amyl alcohol-ether mixture as a golden yeIIow coloured complex with sodium diethyldithiocarbamate. The amount of this formed complex is read at 440 nm. The sodium pyrophosphate is added to prevent interference from iron. Reagents Standard Solution Stock Solution (lOOIlg/ml)Copper sulphate, CuS04 • SHP 0.398 g DistilJed water 1 litre Sulphuric acid, concentrated 0.1 ml Working Standard (1 Ilg/ml): Dilute the stock 1: 100 with distilled water. Procedure o Pipette 3 ml of serum in a test tube. o Add 1 ml of 0.1 N HCl., warm in boiling water bath, stire continuously until the mixure begins to cloud. o Cool and add I.S ml of 6 N HCI and stand for 10 min. o Add 3 ml of 20% trichloroacetic acid, mix weIl and stand for a few minutes. o Centrifuge at 3000 rpm for 10 - IS min. o Remove supernatant and wash precipitate with 3 ml of 5% trichloroacetic acid. o Centrifuge at 3000 rpm for 10 min and remove supernatant. o Mix both the supernatant fluids in another tube. o Add 1 ml of 6% sodium pyrophosphate and 2 ml of ammonia. o Add 1 ml of 0.4% sodium diethyldithiocarbamate and shake well for 2 min with S ml of the amyl alcohol-ether 1: 1 mixture to extract the copper. o Remove the amyl alcohol layer and dry by shaking with a little powder of anhydrous sodium sulphate. o Read at 440 nm or by using violet filter. o Treat S ml of standard (Standard) and distilled water (Blank) separately in the same way as the serum and take readings at 440 nm. Calculation fJg Cu per 100 ml serum:

=

Reading of Unknown Reading of Standard

X 5 X 100 3

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Techniques in Life Sciences =

Reading of Unknown

X 167

Reading of Standard

Clinical Significance The normal range of serum copper is about 75 to 160 f.lg/lOOml and its 90 to 95% is bound to the ,12 globulin ceruloplasmin, the rest to albumin. About 20 -35 mg of ceruloplasmin is present in plasma. The Cu level increases in chronic infections, pregnancy, in pernicious anaemia and cirrhosis of the liver. 3.15. Titan yellow colorimetric Technique for Magnesium Principle Titan yellow gives a red colour with magnesium. It's intensity can be read at 520 nm. Reagents 1 Standard Solution Stock Standard (1 mg/ml Mg): Dissolve 8.458 g of MgCI 2 • 6Hp in distilled water and make upto a litre or 10.094 g of MgNH4P0 4 • 6Hp in 0.1 N HCI and make up to a litre with distilled water. 2 Working Standard (5f.lg/ml Mg): Dilute 1 ml of stock solution to 200 ml with distilled water. 3 Calcium Chloride Solution (0.05 mg Iml CaY: Dissolve 13.88 mg of CaCI 2 in 100 ml of distilled water. Procedure o Pipette 1 ml of serum and add 5 ml of distilled water (Test). o Pipette 2.5 ml of the standard (Standard). o Pipette 2.5 ml of water as blank. o Add to all above tubes, 2 ml of 10% sodium tungstate and 2 ml of 2/3N H2S04. o Centrifuge all above tubes, at 3000 rpm for 10 min. o Pipette 5 ml of supernatant fluid from each tube and add 1 ml of distilled water. o Add 1 ml of 0.05% titan yellow and 2 ml of 4N sodium hydroxide in all tubes. o Add 1 ml of calcium chloride solution and 5 ml of distilled water in all tubes. o Take readings of test and standard soon after red colour developed, against blank at 520 nm or by using a green filter. Calculation mg magnesium per 100ml serum: Reading of Test X 2.5 = Reading of Standard Magnesium in serum ranges from 1.7 to 2.6 mg /l00 ml or 1.4 to 2.4 m.Eq./L. Clinical Significance Increased serum magnesium occurs in renal disease with oliguria, Ii ver disease and diabetic coma. It falls down during malabsorption syndrome, diarrhea, parathyroid tumour removal and aldosteronism conditions.

• ••

DETECTION OF VITAMINS

1

CHEMISTRY AND CLASSIFICATION

Vitamins are the organic substances required by an animal in a minute quantity as the essential growth factors or physiological regulators. Initially, discoverer of vitamins, Hopkins (1912) termed these substances as the accessory food factors as their source of availability to man and other animals was found to be their food. Most of the vitamins are synthesized by the plants and available in the vegetables, fruits and cereals or some of them may be available as the by-products of intestinal micro-organisms. Only vitamin D can be synthesized by an animal in the skin from steroids in the presence of ultraviolet radiation. Some vitamins activate enzyme activity and Florkin (1971), therefore, called them as the biological catalysts. It is now well established that all the vitamins function as the growth factors or physiological regulators and their deficiency may cause fatal diseases like, beribery, scurvy, xerophthalmia, rickets etc. Physicians, generally, advice vitamin-rich diet for an adequate growth of the children.

Classification and Mode of Action There are about 25 vitamins (Fig. & Table 14.1). The vitamins represent heterogenous group of organic substances like, amino acids, proteins, organic acids, esters, alcohols, steroids and quinine etc. They are grouped into six groups and designated as the A, B, C, D, E and K vitamins. Vitamin A, D and K occur in two forms while vitamin B consists of a number of vitamins forming a vitamin B - complex. Vitamins A, D, E and K are fat soluble and remaining vitamins are water - soluble. Vitamin A is fat soluble yellow oil or a crystalline solid and occurs in two forms Vitamin Al and vitamin A 2. Vitamin Al is Retinol and vitamin A2 is 3,4 dehydroretinol or retinol 2. Vitamin A is stored in the liver as an ester forming lipoglycoprotein complex in the lipocytes. It is later on, hydrolysed and retinol molecule bound to the aporetinol binding protein passes through Golgi and

306

Techniques in Life Sciences

Vitamin B, or Thiamin is obtained from outer coat of cereal grains, liver, eggs in an inactive form. The enzyme, thiamin diphosphotransferase converts thiamin into its active form, thiamin diphosphate in the liver and brain. Vitamin B2 or riboflavin or iactoflavin. It is isolated from vegetables (riboflavin) and milk (lactoflavin) as a yellow water soluble pigment. Active riboflavin is flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) formed from ATP-dependent phosphorylation of riboflavin. Vitamin B3 is pantothenic acid occurring everywhere in animal tissues. Active form of vitamin B3 is the coenzyme A (Co A) and the acyl carrier protein (ACP) converted from pantothenic acid by ATPdependent phosphorylation in intestine. Vitamin B4 is the folic acid obtained from leafy vegetables and lemon, banana, strawberries like - fruits and stored in animal liver and kidney. Active hormone is tetrahydroloate (H4 folate) formed in intestine after reduction carried out by enzymes. Vitamin B5 is nicotinic acid or niacin. It is widely distributed in plant and animal tissues. The active vitamin B5 is the nicotinamide derived from pyridine after undergoing deamidation to nicotinate. It is required for synthesis of NAD and NADP Nicotinate is converted to NAD and NADP which function as the s;:oenzymes I and II and carry our oxidation reactions. Vitamin B6 is a group of three vitamins, pyridoxine, pyridoxal and pyridoxamine. They occur in various plants and animals, absorbed by intestine and converted into pyridoxal phosphate by the enzyme pyridoxal kinase. and transported in the plasma. It acts as a coenzyme and takes part in glycogenolysis. Biotin or vitamin H is an imidazole derivative distributed in food plants and animal products. a-biotin is active hormone formed mostly by intestinal bacteria. It functions as the coenzymes pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase and betamethylcrotonyl-CoA carboxylase. Vitamin B12 or cyanocobalamin is synthetized in microorganisms and may be stored in contaminated plants and liver of the animals in the form of methy lcobalamin, adenosy lcobalamin and hydroxocobalamin. It acts as coenzymes like methylcobalamin and deoxyadenosylcobalamin. Vitamin C or ascorbic acid is obtained from citrus fruits and is, in fact, a hexose -sugar derivative. It acts as a donor of reducing equivalents or as a reducing agent. Dehydroascorbic acid is the true vitamin C. It hydrolyses proline in collagen synthesis. Vitamin D includes 5 forms of steroids as D" D 2, D 3, D4 and D5derived from ergosterol or 7-dehydrocholesterol (found in human skin) under radiation. Three types - D, (Calciferol), D2 (Ergocalciferol) and D3 (Cholecalciferol) are equally active substances. Best source is the cod liver oil. Active vitamin D consists of two OH groups. Vitamin D accelerates rate of Ca ++ and P043- ions through the intestine wall. Vitamin E consists of 5 compounds- a-, ~-, y-, 0- and E tocopherols but only atocopherol is found to be a fully-biologically active vitamin E molecule. It is found in plants and animal tissues but occurs predominantly in wheat germ oil in a- and ~- forms while soyabean oil contains &--tocopherol. It acts as a natural antioxidant breaking radical chain reactions during peroxidation of polyunsaturated fatty acids of phospholipids in the cellular (mostly, mitochondria, endoplasmic reticulum and Golgi) membranes.

307

Detection of Vitamins

Nomenclature, chemical nature, source and deficiency effects of the vitamins are briefly mentioned below:

Vitamin ChemicaINature A:Aj

Az

Table 14.1 Vitamins at a glance Deficiency Effects Source

u-carotene alcohol}

fish, liver, egg, milk,

Dryness and keratosis

~-carotene alcohol}

butter, carrots, tomatoes

of skin and cornea, Night blindness

(Diterpenoid Retinol) B: B j

Thiamin

cereals, rice, liver, egg, milk, Beri-beri wheat bran, green vegetable sea food, ground nut, yeast

B2

B3 B4

Bs

Riboflavin

Pantothenic acid

Folic acid

Nicotinic acid (Niacin)

B6

Pyridoxine

Biotin

H

Bj2 Cyanocobalamin C

Ascorbic acid

Liver, cheese, milk, eggs,

Dermatitis, cheilosis,

meat, soyabean, vegetables

premature ageing

Eggs, meat, milk, wheat,

Dermatitis, depression

Peanuts, liver, kidney, yeast

Burning eye sensation

Green leafy vegetables, fruit

Macrocyclic Anaemia

Beef, yeast, soyabean

(RBC deficiency)

Meat, liver, egg, milk, coffee Pellagra (mental dispotatoes, grains, pulses.

order) red skin spots

Yeast, cereals, milk, eggs,

severe dermatitis,

liver, meat, wheat - germ

mental disorder.

Liver, milk, yeast

Dermatitis, hair loss

Meat, fish, liver, egg, milk,

ernicious anaemia,

fruits

Blood formation loss

Citrus fruits,

Scurvy, dental

green vegetables

disease, fatigue.

Calciferol,

Fish liver oil, animal fat,

Rickets, skeletal

Cholecalciferol

butter

deformities.

Tocopherol

Wheat germ oil, palm oil,

Antisterility,

soyabean oil, cotton seed oil

Leucocyte increase.

Naphthoquinone

cereals, leafy vegetables

Haemorrhages

Kj

Phylloquinone

Alfalfa, carrot, cabbage

(Coagulation factor)

Kz

Famoquinone

D: D j D2 E

K

Bacteria and putrefied fish

308

Techniques in Life Sciences

.I1-CHa

CHI'

CHi

l)-CH=CH-~=CH-CH=CHJ=CHCHaOH

/". .

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.h"

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HO~

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I

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I

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I

(C!W.-CH-tcHzl.CH- I 00. Calculate the concentration of viable and non-viable cells and the percentage of the viable cells using the equation given below: Concentration of viable cells (cells/ml) =A xC XD. Concentration of non-viable cells (cells/ml) =B x C x D. Total number of viable cells =concentration of viable cells x volume. Total number of cells = number of viable + number of dead cells. Percentage Viability =(No. of viable cells x 100) -:- Total No of cells.

o

o o o o

o o

Animal Cell and Tissue Culture

415

Where, A -Mean of viable cells counted, i.e., Total viable cells counted 7 No. of squares. B - Mean of non-viable cells counted, i.e., Total non-viable cells counted 7 No of squares C -Dilution factor and D -Correction factor (provided by haemocytometer manufacturer. Note 1. Use Trypan Blue carefully as it is toxic and potent carcinogen. Do not breathe the vapours. 2. The correction factor of 104 converts 0.1 mm 3 to 1 ml (0.1 mm 3 = 1 mm2 x 0.1 mm). 2.6. MTT (Yellow Tetrazolium Salt) Cell Proliferation Assay Aim Measurement of cell viability and proliferation forms the basis for numerous in vitro assays of a cell population's response to external factors. The reduction of tetrazolium salts is now widely accepted as a reliable way to examine cell proliferation. The yellow tetrazolium MIT (3-(4,5-dimethylthiazolyl-2) - 2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric method. The MIT cell proliferation assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. For each cell type, the linear relationship between cell number and signal produced is to be established for allowing an accurate quantification of changes in the rate of cell proliferation. Material Required 1. MIT Stock solution - 5 mg/ml dissolved in PBS. 2. PBS without Ca++ and Mg++ 3. 0.25% TrypsinlEDTA in HBSS withoutCa++ and Mg++ 4. DMSO, 5. Microtiter plate reader with 650 and 570 nm filters, 6. Multi-channel pipettes etc. Procedure D Harvest suspension cells by centrifugation. Adherent cells should be released from their substrate by trypsinization or scraping. D Resuspend cells at 1 x 106 per ml. D Prepare serial dilutions of cells in culture medium from 1 x 106 to 1 X 103 cells per ml. D Plate out in triplicate, 100 f.11 of the dilutions into wells of a microtiter plate. D Include three control wells of medium alone to provide the blanks for absorbance readings. D Incubate the cells under conditions appropriate for the cell lines for 6 to 48 h. Incubate for 12 h to overnight. D Add 10 f.11 of MIT stock solution to each well, including controls. D Incubate for 2 to 4 h until purple precipitate is visible. D Remove the medium carefully without disturbing the cells. D Add 100 J..I:I DMSO and pipette up and down to dissolve the crystals.

416 D D

Techniques in Life Sciences Leave at room temperature in the dark for 5 - 10 min. Record absorbance at 570 nm.

Result Absorbance values that are lower than the control cells indicate a reduction in the rate of cell proliferation. Conversely, a higher absorbance rate indicates an increase in cell proliferation.

2.7.

Cryo-preservation of Cell Lines

Aim Cryopreservation of cell lines can be carried out at -80°C electric freezer and liquid nitrogen or a programmable rate controlled freezers.

Materials Required 1. Freeze medium - 70% basal medium, 20% FCS, 10% DMDO or Glycerol. 2. 3. 4. 5. 6.

70% ethanol. PBS without Ca++ and Mg++ 0.25% TrypsinlEDTA in HBSS without Ca++ and Mg++ Cryo-preservative liquid - Dimethyl sulphoxide (DMSO), Cell freezing device (Nalgene Mr. Frosty), Haemocytometer, liquid nitrogen container and Deep Freezer (-80°C).

Procedure D

Examine under microscope density of cells in culture and confirm any kind of microbial disinfection. D Take adherent and semi-adherent cells into suspension using trypsinlEDTA and resuspend in a volume of fresh medium at least equivalent to the volume of trypsin. Suspension cell lines can be used directly. D Remove a small aliquot of cells (100 - 200) and perform a cell count by haemocytometer. D Centrifuge the remaining culture at 150g for 5 min. D Re-suspend cells at a concentration of 2-4 x 106 cells per ml in freeze medium. D Pipette 1 ml of aliquots of cells into cryoprotective ampules (containing DMSO -serum (90: 10) in medium) that have been labeled with the cell line name, passage number, cell concentration and date. D Place ampules inside a passive freezer e.g., Nalgene Mr. Frosty. Fill freezer with isopropyl alcohol and place at -80°C overnight. D Frozen ampules should be transferred to the vapour phase of a liquid nitrogen storage vessel and the locations recorded. Note - 1. For HL60 like cell lines DMSO is not a good cryoprotectant and therefore, it is to be replaced by glycerol. 2. Cultures should be healthy and in log phase of growth. It can be achieved by selecting the pre-cunfluent cultures (below maximum cell density) and by changing the culture medium 24 h prior to freezing. 3. Place the appropriate volume and cell number into cryopreservation ampules -'usually 2 x 105 to 5 x 10 6 cells / ml ampUles.

41 7

Animal Cell and Tissue Culture

2.8. Aim

4. Plastic ampules with external silicone seals function best when kept above liquid nitrogen temperature i. e., in the vapour phase. S. Super insulate liquid nitrogen containers or tanks are more perfect than the conventional thermos bottles. 6. Thawing should be performed as rapidly as possible without letting the temperature exceed 37°C. The best way to warm sample is by immersion in a 37°C water bath with gentle agitation. Lyophilisation and Freeze - Dry Technique for Cellffissue Preservation

This method is used to preserve a stock of microorganisms like bacteria and cell tissue cultures at a large scale. The material can be freezed, dehydrated and stored for a longer duration (even more than 30 years). Procedure o A dense cell suspension is placed in small plastic vials and frozen at - 60 to -78°C. o The cells are then connected to a high vacuum line. The ice present in a frozen suspension sublimes under the vacuum i.e., evaporates without first going through a liquid water phase. This results in dehydration of the bacteria or cells with a minimum of damage to delicate cell cultures. o The vials are then sealed off under a vacuum and stored in a refrigerator. o Small cotton plugged vials containing frozen suspensions of bacteria are placed in the "lyophilization apparatus" consisting a glass-flask, which is attached to a con~enser. The condenser is connected to a high vacuum pump. The bacteria/cells become desiccated as the ice in the frozen suspension sublimes directly to water vapour. The vapour is trapped on the cold surface of the condenser, thereby preventing it from entering the Seal (By flaming open end )

Asbestos packing provides insulation during sealing

Inner vial (cotton plugged) ~~+-_Dry

ice & alcohol

containging lyophilized specimen

Outer glass tube Vials containing frozen (bacterial) cell line suspension

Dewar Flask

Fig. 18.2 Lyophilization process for preservation of cultures.

418

Techniques in Life Sciences

vacuum line and contaminating with the pump oil. After desiccation of cultures, the vials are removed and each is placed in a larger tube. After insulating the vial with a plug of glass wool packing, the outer tube is hermetically sealed under a vacuum by means of torch availed from ATCC (American Type Culture Collection). Note - 1. Hundreds of lyophilized cultures can be stored in a minimum space. 2. Lyophilized cultures are revived by opening the vials, adding liquid medium and transferring the rehydrated culture to a suitable growth medium.

o o

2.9. Revival of Resuscitation of Frozen Cell Lines Aim Make cell cultures, such as, NCCS India, ATCC USA, or ECACC UK are supplied to various laboratories in frozen state. Similarly, cell lines are, often kept in frozen state below 196°C'in liquid nitrogen or deep freezers . Prior to use, the frozen cells should be thawed and put into culture in such a way that viability of the culture should be maintained, and the culture should recover quickly. A due care should be taken at this time that the DMSO like cryoprotectants are toxic to the cells above 4°C. Material Required Culture Medium like RPMI, DMEM etc can be commercially available in powder form, which can be reconstitutesI in autoclaved milli-Q water according to the manufacturers' details or be obtained in liquid form (Sigma, Aldrich etc.). Procedure Collect ampule of cells from liquid nitrogen storage and transfer to laboratory in a sealed container. o Remove ampule from a container and place in a water bath at 37°C for mammalian cells or at appropriate temperature for other group of animal cells by submerging only a lower half of an ampule and leave for 2 min or until a small amount of ice remains in the vial and transfer to laminar flow hood. o Wipe out the outside of an ampule with a tissue moistened with 70% ethanol and hold tissue paper over ampule to open the lid. o Transfer the cells slowly with a help of pipette in to a prewarmed growth medium to dilute out the DMSO in a flask labeled appropriately. o Incubate at appropriate temperature for species and concentration of CO2 in atmosphere. o Examine the cells under inverted microscope having phase-contrast attachment after 24 h and subculture as necessary. Note: 1. Do not use an incubator to thaw cell cultures otherwise, viability may get lost. 2. If CO2 incubator is not available, then gas the flasks for 2 min with 5% CO2 in 95% air filtered through a 0.25 m filter.

o

2.10. Detection of Bacterial and Fungal Contamination Aim The low-level bacterial and fungal contamination can not be visualized by nacked eyes. It is therefore, essential to sterilize by incubation of cell cultures in microbiological broth

Animal Cell and Tissue Culture

419

prior to conduct any experiment or to proceed for cell banking. Material Required 1. Soybean Casein Digest (Tryptone Soya Broth, TSB) 15 ml aliquots or TSB powder, 2. Fluid Thioglycollate medium 20 ml of aliquots (TGM), 3. Bacillus subtilis, Candida albicans, Clostridium sporogenes, (available from the National Collection of Type Cultures (NCTC), u.K. Procedure o Culture cell lines in the absence of antibiotics for two passages prior to testing. o Bring attached cells in to suspension by using cell scraper. Inoculate 2 X TGM and 2 X TSB with 1.5 ml of test sample. o Inoculate 2 X TGM and 2 X TSB with 0.1 ml of each Bacillus subtilis, Candida albicans, Clostridium sporogenes containing 100 colony forming unit (cfu) separately. o Leave 2 (TGM) and 2 (TSB) un-inoculated as negative controls. o Incubate broths as follows: a) For TSB, inoculate one broth of each pair at 32°C, the other at 22°C, for 14 days. b) For TGM, inoculate one broth of each pair at 32°C, the other at 22°C, for 14 days. c) For the TGM, inoculated with C. sporogenes incubate at 32°C for 14 days. o Examine test and control broths for turbidity after 14 days. Result All inoculated cultures with each, Bacillus subtilis, Candida albicans and Clostridium sporogenes show infection under microscope. Similarly, Test broths containing bacterial or fungi show turbidity. The control broths, on the contrary, do not show infection of bacteria and fungi after a period of 14 days. 2.11. Detection of Mycoplasma Contamination by Hoechst Staining Technique Principle Hoechst fluorescent staining technique stains mycoplasma extra-nuclear DNA besides the nuclei of the cells of infected culture while the cells of un infected culture show only fluorescing nuclei against a dark back ground. Procedure o Sterilize 4 coverslips in a hot oven at 180°C for 2 h or by immersing in 70% ethanol and drying on a blue Bursen flame. Place a cover-slip in each culture dish of 35 mm size. Add 2 x 104 cells in 2 ml of antibiotic-free growth medium to each tissue culture dish (Preparation of Vero indicator cells). o Incubate at 37°C in 5% CO2 for 2 - 24 h to allow the cells to adhere to the cover-slips. o Bring attached test cell lines into suspension using a cell scraper. Suspension cell lines can be tested directly. Remove 1 ml of culture supernatant from duplicate dishes and add 1 ml of test samp Ie to each. Inoculate 2 dishes with 100 cfu M. hyorhinis and 2 with 100 cfu M. orale (available from the national collection of type cultures (NCTC, U.K.). o Leave duplic;ate tissue culture dishes un-inoculated as negative controls. o Incubate dishes at 37°C in 5% CO2 for 1 - 3 days.

o

o o

o

420

o

Techniques in Life Sciences

After 1 day observe one dish from each pair for bacterial or fungal infection. If contaminated, discard immediately. Leave the remaining dish of each pair for a further period of 2 days. o Fix cells to cover-slip by adding a minimum of 2 ml of freshly prepared fixative (1:3 glacial acetic acid and absolute methanol) to the tissue culture dish and leave for 5 min. o Decant used fixative to toxic waste bottle. Add another 2 ml aliquot of fixative to coverslip and leave for 5 min. o Air dry cover-slip by resting it against the tissue culture dish for I h. o Replace cover-slip in dish and add a 2 ml of Hoechst stain. Leave for 5 min. Shield from direct light by aluminium foil. o Decant used staining solution to toxic waste. o Add 1 drop of mount ant to a pre-labeled microscope slide and place cover-slip over a drop of mountant. Cover the slide with aluminium foil and leave for 15-30 min at room temperature. o Observe the slide under ultra violet Epi-Fluorescent microscope at x 1000. Result Samples infected with mycoplasma show fluorescing nuclei as well as extra nuclear fluorescence of mycoplasma DNA as small cocci or filaments while unaffected samples show fluorescing nuclei against dark background only. 2.12 Harvesting and Plating Bone Marrow-Derived Microphages Material Required 2 month old C57BL6 male mice, CO 2, Sterile 50 ml polypropylene conical centrifuge tubes, Sterile syringes, 10 mL, Sterile needles, 18 and 22 gauge, Sterile pipettes, 100 mm non-tissue culture treated petri dishes, Sterile DPBS, Bone marrow-derived macrophage growth medium 1, 70% Ethanol, 100 mm treated tissue culture Nunc dishes, Blunt end scissor, Mayo scissor or sharp dissecting scissor, Forceps , Tissue culture hood. For the following procedure, use items that are sterile or have been sprayed with 70% ethanol. Procedure o Immediately before surgery, sacrifice mice with CO2, o Prepare one mouse at a time on a clean sheet of absorbent paper. o Spray all external areas of the mouse with 70% ethanol. o Using a blunt end scissor, make an incision 1 inch vertically from umbilical region to anterior region. o Extend this incision along the medial aspect of both rear appendages. o Gently pull the skin downward below the heels to expose the muscles, etc. o Using a sharp scissor, dissect tibias and femurs from surrounding muscles and tendons. Place the tibias and femurs into a 50 ml polypropylene tube containing 4°C DPBS. o In a tissue culture hood, place the tibias and femurs on a non-tissue culture treatedPetri

Animal Cell and Tissue Culture

D D

D D D D D D D D D D D D D D

421

dish and spray with 70% ethanol. Using a sharp scissor, remove excess tissue, knee and heel. With an 18 gauge needle, fill a 10 ml syringe with - 8.5 ml bone marrow derived macrophage growth medium then replace the 18 gauge with a 22 gauge needle. (Syringes may be prepared ahead of time) Drill the needle into the end (previous knee junction) of the femur or into the end (previous ankle junction) of the tibia. Flush 2 ml of the BMDMGM I through the femur and another 2 ml through the tibia onto a new non-tissue culture treated petri dish. Suspend cells and medium 1 x with a 22 gauge needle and a 10 ml syringe. Place the cells into a 50 ml polypropylene tube. Divide the cells per mouse among 6 - 10 cm non-tissue culture treated petri dishes and bring the volume to 7 ml with BMDMGMI per dish. Maintain cells in a humidified 37°C incubator. On day 4, wash the cells with 7 ml RPM!. Add 7 ml fresh BMDMGMI and place back in incubator. On day 6, aspirate BMDMGMl, wash the cells I x with 5 ml 37°C DPBS, add 5 ml of 37°C DPBS plus I mM EDTA and incubate at 37°C for - 5 minutes. Pipette cells off the bottom of the dish using a 10 mL pipette and place in a 50 ml polypropylene tube containing 10 ml BMDMGMI. Pellet the cells by spinning at 1500 RPM for 5 min in table top centrifuge. Aspirate the supernatant and suspend cells in 1 ml fresh BMDMGMI per mouse. Count cells by making a 10 fold dilution (100 ~l cell suspension plus 900 ~IDPBS). Plate cell density as outlined below in 37°C BMDMGMI. 6 well dish: I x 106 13 ml medium 60 mm dish: 3 x 10615 ml medium 100 mm dish: 5 x 106 /7 ml medium 150 cm 2 flask: I x 107/20 ml medium.

2.13 Isolation and Growth of Mouse Primary Myoblasts

, \

Primary mouse myoblasts can be purified, stably transduced, and grown extensively in vitro. The transplantation of such cells can thus be used as a method of gene delivery. Primary cultures derived from skeletal muscle consist mainly of myoblasts and fibroblasts and the following protocol describes the purification of myogenic cells from such a mixed population. During the initial week or two of CUlturing, selective growth and passaging conditions allow the myoblasts to become the dominant cell type, and after two to three weeks in culture, nearly 100% of the cells stain positive for the myoblast specific protein desmin. Primary myoblasts can be isolated from mice of any age, but isolation from neonatal mice gives a greater yield of myogenic cells. Material Required 70% ethanol in a squirt bottle, Sterile phosphate buffered saline (PBS), Collagenase/dispase/CaCl2 solution,

422

Techniques in Life Sciences

F-lO-based primary myoblast growth medium, F-I0IDMEM-based primary myoblast growth medium~ Fusion medium, Neonatal mice, preferably 1-3 days old, Sharp curved surgical scissors, 2 pairs of fine forceps Low power stereo dissecting microscope, Sterile razor blade, 80 m nylon mesh (e.g. Nitex; Tetko, Inc.; Monterey Park, CA) Small sterile funnel, Collagen-coated tissue culture dishes: 35 mm, 60 mm, 100 mm, and 150 mm sizes, Humidified 37°C, 5% CO 2 incubator Inverted microscope Reagents and Solutions Collagen-coated tissue culture dishes Add 1 ml concentrated acetic acid to 179 ml distilled water, Sterile filter through 0.2 ~m filtration unit, Add 20 ml 0.1 % calf skin collagen in 0.1 N acetic acid, Incubate in plastic tissue culture dishes overnight at room temperature, Remove collagen solution (can be reused; store at 4°C), Rinse dishes well with sterile distilled water, allow to dry Store at room temperature for at least 6 months CollagenaseidispaseiCaCl2 solution 1.5 U/ml collagenase D (Boehringer Mannheim Corp.) from -200C frozen aliquot 2.4 U/ml dispase II (Boehringer Mannheim Corp.) from -200C frozen aliquot 2.5 mMCaCl 2 Use immediately. F-JO-based primary myoblast growth medium 400 ml Hams F-l 0 nutrient mixture (GIBCO BRL) 100 ml fetal calf serum (HyClone Laboratories, Inc.) (20% final) 50 ~l of 25 ~g/ml basic fibroblast growth factor (human; Promega Corp.) in 0.5% bovine serum albuminIPBS; freeze aliquots only once at -20'C (2.5 nglml final) 5 ml 100x penicillin/streptomycin (GIBCO BRL; final concentration is 100 U/ml and 100 ~g/ml, respectively). Store at 4°C for at least 1 month F-JOIDMEM-based primary myoblast growth medium Same as above but use 200 ml F-lO and 200 ml Dulbeccos Modified Eagle Medium (DMEM; GIBCO BRL) in place of the 400 ml F-lO. Fusion medium 95 mlDMEM 5 ml horse serum (HyClone) (5% final) 1 mllOOx penicillin/streptomycin solution (above)

I f

Animal Cell and TIssue Culture

423

Store at 4"C for at least I month Phosphate buffered saline (PBS), lOx stock 2.0 gKCI 2.0 g KH2P0 4 80.0 g NaCI 11.4 g Na2HP0 4 (anhydrous) Dissolve Na2HP04 in 200 ml double distilled water with stirring and heating Dissolve remaining three salts in 700 ml double distilled water; combine both solutions Adjust pH to 7.4 with 5N NaOH Adjust volume to I liter and autoclave or filter. Store at room temperature. Procedure (A) Isolate limb muscle o Sacrifice 1-5 neonatal mice by decapitation or CO2 inhalation. o Rinse the limbs with 70% ethanol and remove them with sterile scissors. Dissect the muscle away from the skin and bone with sterile forceps. Dissection is easier if done under a stereo dissecting microscope. Store the muscle tissue in a culture dish on ice in a drop of sterile PBS as successive limbs are being processed, maintaining sterility in the accumulated tissue. (B) Dissociate muscle cells o Add enough PBS to keep the tissue moist, and mince to a slurry with razor blades in the culture dish. Do this and all subsequent steps in a sterile tissue culture hood. o Add approximately 2 ml of collagenase/dispase/CaCl2 solution per gram of tissue and continue mincing for several minutes. For the amount of tissue obtainedfrom 1-5 neonatal pups, 0.5 ml of the enzyme solution is usually sufficient. o Transfer the minced tissue to a sterile tube and incubate at 37°C until the mixture is a fine slurry, usually about 20 min. Several times during the incubation, gently triturate with a plastic pipette to break up clumps. o If desired, the slurry can be filtered through a piece of 80 ~m nylon mesh in a sterile funnel to remove large pieces of tissue. o Centrifuge the cells for 5 min at 350 x g. Resuspend the pellet in 2-4 ml of F-IO-based primary myoblast growth medium depending on the amount of tissue processed, and plate in a 35-60 mm collagen coated culture dish. It is common to see a great deal of debris and very few recognizable cells; however, after two days, many cells will be evident and the debris will be rinsed away during the first change of medium. (C) Enrich/or myoblasts o Incubate in a 37°C 5% CO2 incubator and change the medium every 2 days, using F-lObased primary myoblast growth medium. Do not leave cells in the same culture dish for more than 5 days, regardless of cell density. Primary myoblasts grow best when dense. Do not grow them at less than approximately

•

424

o

O·

o

o

o

Techniques in Life Sciences 10% confluence, but also do not allow them to become too crowded, or else they may start to differentiate or die. Split them at no more than 1:5 dilution. The F-JO-based primary myoblast growth medium gives myoblasts a growth advantage over fibroblasts. When the cells are ready to be split, remove the cells from the dish using PBS with no trypsin or EDTA. This is accomplished by aspirating off the growth medium, rinsing the dish with PBS, leaving a small amount of PBS in the dish, and hitting the dish very firmly in a sideways fashion against the edge of a table top to dislodge the cells. It can take several minutes of incubation in the PBS before the cells can be knocked off of the tissue culture plastic. However, the myoblasts will freely come off during this treatment and many fibroblasts will be left behind. Preplate the cells for 15 min on a collagen coated dish before moving the cells remaining in suspension to a new collagen coated dish. This tends to leave behind cells that stick quickly, which are predominantly fibroblasts. To avoid leaving myoblasts behind, swirl the liquid in the dish, tilt it towards the pipette, andjostle the dishfrom side to side while slowly removing the cell suspension. Failure to do this can result in a significant number of myoblasts trapped in the meniscus. Repeat steps 9 and 10 for the initial week of culture expansion or until most of the fibroblasts are gone from the culture. Fibroblasts tend to be very flat when grown on collagen, whereas myoblasts are more compact and much smaller in diameter. Myoblasts can also be identified by immunofluorescent staining for desmin. After the fibroblasts are no longer visible in the culture, the medium can be changed to FIO/DMEM-based primary myoblast growth medium. This medium allows myoblasts to grow faster and to higher densities. However, it also removes the selective growth advantage of myoblasts over fibroblasts, and thus should not be used until after the fibroblasts are gone. This medium can be changed every three days instead of every two, but care should still be taken to avoid overgrowth of cells. Primary myoblasts can be frozen for storage using standard cell culture protocols. After approximately one week ofgrowth, the myoblasts usually go through a crisis period during which a significantfraction of the population (sometimes the majority) dies. This tends to occur at the time that the fibroblasts have disappeared from the culture, although a direct correlation has not been confirmed in our laboratory. The remaining myoblasts soon repopulate and have a remarkable proliferative ability (up to JOO doublings) but still retain the ability to differentiate under the proper conditions. Confirm ability to differentiate in culture If desired, the potential of the myoblasts to differentiate in culture can be assessed by replacing the medium with fusion medium. The medium must be changed daily. Start with an approximately 30-50% confluent dish of cells. Within several days to one week, large multinucleated myotubes should be quite obvious. After approximately one week in fusion medium, the myotubes can sometimes be observed to twitch randomly as the contractile machinery assembles.

Animal Cell and Tissue Culture 3 3.1

425

CELLCULTUREAND~OCYTOCHE~TRY

Light Microscopic Immunocytochemistry of the cells in Tissue Culture

Principle Immunofluorescence is the preferred detection method for cultured cells; it is much less prone to artifactual labeling than enzyme-linked techniques and is more quantitative. Enzyme-linked methods can be very sensitive but have very narrow dynamic ranges, with poor differentiation of amino acid levels in different cell types. Detection for glutamate, aspartate, glutamine, glycine, like amino acids are not all-or-none procedures as cells will contain varied levels that may discriminate functional cell types. The most quantitative method is silver-enhancement.

Basic Working Reagents 1 2 3 4 5 6 7 8 9 10

Prepare a 100X dilution of a selected Series 100 Antiserum or Prepare a lOX dilution of a selected SKlOO Explorer Kit Antiserum PB = phosphate buffer, pH 7.4 PBS =osmotically proper PB saline for cells, pH 7.4 Triton X-l00 1% GSPBS = 1% goat serum in PBS 0.5% NaBH4 in deionized water. 2° Ab: fluorophore conjugated anti-rabbit IgG Mounting medium 2% Na azide in glycerol

Procedure Fixation: Quantitative trapping of free amino acids requires a bifunctional linker such as glutaraldehyde. Optimal trapping will occur with a standard 0.5%-2.5% glutaraldehyde/ 1% paraformaldehyde mixture. Depending on the amount of native antigen in cells, even lower levels of glutaraldehyde may be used along with higher paraformaldehyde. The increased path length through cultured cells often permits low levels of glutaraldehyde to yield excellent signals. Fix at 4 hrs-overnight at 4°C. Wash 10 min 3X in PBS. Reduce autofluorescence and glutaraldehyde induced fluorescence: Wash 1 min in 0.5% NaBH4 in deionized water. Note: This step could mechanically damage cells if left too long. This step is imperative if FITC or or any other green dominant fluorophore is used, but is less critical (though still beneficial) for long-wavelength fluorophores such as rhodamine or Cy3tm (Amersham). Wash 10 min 3X in PBS. Carefully dry around the well with a lint-free wipe. Primary Blocking: Blocking may be needed with cultured cells. If desired, block with 50 III of 2% goat serum / 0.1 % Triton X-lOO PBS for 1 h. Apply antiserum: Remove blocking agent with a pipette and replace with 50 III of IgG diluted with GSPBS + 0.1 % Triton X-lOO. Incubate overnight at room temperature or 4°C if desired. The recommended dilutions should work well for most amino acids, but should signals seem weak, 1.5-2X stronger dilutions be used. However, if much higher

426

Techniques in Life Sciences concentrations are required it is very likely that (a) there is simply to little amino acid to detect; (b) fixation losses have been excessive; (c) the detection method is insensitive. Wash 10 min 3X in PBS. Carefully dry around the well. Secondary Blocking: If desired, block with 50 microliters of 2% GSPBS 10.1 % Triton X-IOO. Secondary antiserum: Incubate 30-45 min in secondary goat anti-rabbit IgG diluted as recommended by the manufacturer and supplemented with 0.1 % Triton X-IOO. Wash 10 min 3X in PBS plus 5 min X4 in deionized water. Dry around well. , Coverslip: Fill well with antifade mountant such as IMMUMOUNTtm, DABCOtm or, for FITC, 1 part PBS, 3 parts glycerol, plus 2.5% Na azide. A coverslip can be placed over the well if desired. If the cells are to be used for confocal microscopy, the coverslip containing the cells is carefully removed with a razor blade and mounted on a slide with anti fade mountant. Silver intensified specimens may be permanently mounted in standard media. Storage : Store mc frozen. Other fluorescent labels may be stored at 4°C or frozen as appropriate for the fluorescent tag. Silver images are archival.

Note: 1. As basic vehicle, use a PBS formulation osmotically proper for cultured cells, as they lack structural support from other cells or an embedding matrix. Care must be taken to ensure that liquid exchanges do not wash the cells off the dish. This can occur at any point in the procedure from fixation to coverslipping. Gentle fluid transfer is sufficient for washes. Composition of the fixation buffer can also impact adhesion if it strongly buffers divalent cations. Do not allow the well to dry out at any time as this can raise backgrounds to unacceptable levels. Triton X-IOO (0.1 %) is added to the primary antiserum diluent to enhance antibody penetration. While it may not be necessary for all IgGs, it tends to provide a very constant level of performance across preparations and certainly does not attenuate signals. 2. Culture dishes labeled with fluorophores can be coverslipped in an anti-fade DABCOtm, IMMUMOUNTtm mountant and stored under conditions optimal for various fluorophores (FITC< O°C, TRITC 4°C). Background signals may be slightly higher with in vitro experinments compared to post-embedding sections: Agents that may normally bind antisera non-specifically may be eliminated by embedding and etching procedures; coverglasses in culture systems coated with adhesion agents may increase background; and, with in vitro labeling, the specimen is 5-10 11m thick compared to 500 nm less for most post-embedding sections. 3.2 Quick Immunostaining Method for cells in culture Procedure Starting material: grow cells on 18 x 18 mm square or 19 mm circular coverslips in

427

Animal Cell and TIssue Culture

o

o o o

o

o o

o

tissue culture dishes Rinse cells with PBS (optional step). Fix as desired in paraformaldehyde or methanol fixation and rinse well with PBS. Permeabilize with 1% Triton X-100 in PBS for 10 minutes at room temperature. Rinse well with PBS. Set up humidified chamber (box with a lid and water-saturated tissue in it; bottom lined with parafilm) and place coverslips as desired (e.g. to use low solution volumes, in the range of 20-40 I.tI. Pipette antibody directly onto parafilm and carefully place coverslip upside down, i.e. with cells facing down, on top of the drop of solution; if using solution volumes greater than 50 Ill, then place the coverslip on the parafilm with the cells facing up and pipette the antibody directly onto it. Before adding antibody, however, block non-specific antibody binding sites by incubating cells for 10 min in blocking buffer (PBS + 0.1 % Tween + 1% serum). After this step, incubate cells with primary antibodies diluted in blocking buffer for 35 mins to I hour. try several dilutions (low of 1: 100 to high of 1: 1000, and finalize appropriate dilution Transfer coverslips to 6-well plates for PBS washes (3 x 10 min). When washes are done, transfer back to humidified chamber for secondary antibody incubation). Incubate coverslips for 35 mins to 1 hour with the appropriate secondary antibodies diluted in blocking buffer, and wash 3 X 10 minutes with PBS. We use fluorophore-conjugated secondary antibodies from Jackson Immunochemicals. Transfer back to the 6-well plate for final PBS washes (3 x 10 min), and then stain with DAPI if desired (I: 15,000 dilution in water of a 5 mg/ml stock). A good RNA stain, which fluoresces in the red channel, is Pyronin Y (l: 10,000 dilution in water of a 10 mM stock; must stain with DAPI first). Wash well with PBS. Mount the cells for immunofluorescence analysis with MOWIOUDabco. The coverslips must be sealed with nail varnish. Slides can be stored in the fridge or freezer until analyzed by microscopy.

3.3 Double Antibody Conjugation Method of Immunocytochemistry Solutions Required: PHEMbuffer: 25 mMHEPES 60 mM PIPES 2 mM MgCl2 (Add in this order.) Antifade: 1 ml 1 mg p-phenylene diamine hydrochloride Dissolve in 0.1 ml lOx PBS (20 min at RT) Add 0.9 ml 100% glycerol Keep covered at all times and no vortexing. If it turns brown, it's no good. Aliquot and store at -70°C.

Procedure A. Plating:

o

\

To sterilize glass coverslips, dip in ethanol and flame.

10 mMEGTA pH = 6.9

428

o

o o o o o o o o o o o o o o

Techniques in Life Sciences Use 22x22x1 mm3 coverslips and put ~hem in 6-well plates. Seed 100,000 cells per well overnight and fix the next day. B. Fixation: Remove the media and rinse once with PBS. Remove the PBS and immediately add _20DC methanol. (Do not allow the cells to dry.) Put the plate in a _20DC freezer for 5 min. Remove the methanol and add PHEM buffer. Fixed cells are kept at 4 DC in PHEM. C. Antibody incubation: Block with appropriate sera (2.5 to 5%) in PHEM buffer for 1 hr with gentle rocking. Add primary antibody to the blocking buffer and incubate for I hr with gentle rocking. Remove and wash 4 x 10 min with PHEM buffer. Add secondary antibody in PHEM buffer with sera and incubate for 30 min with gentle rocking. Remove and wash 4 x 10 min with PHEM buffer. D. Mounting: Pick up coverslip with forceps and drain away excess buffer (can gently aspirate if desired). Put -20 III "antifade" on slide and gently lay coverslip on top. After removing excess antifade, either by blotting with Kimwipe or aspirating, seal with Sally Hansen clear nail polish. (This brand supposedly works better than others.) KEEP IN THE DARK AT ALL TIMES. Store in - 20 DC in freezer.

ANNEXURE Immunohistochemistry on fixed, paraffin-embedded sections Principle Immunostaining on formalin-fixed, paraffin embedded sections has been revolutionized in 1991 by the discovery of heat-mediated retrieval (Antigen Retrieval, AR) of immunoreactivity. This method is now widely used and applies to the detection of the overwhelming majority of antigens, with few exceptions for which enzymatic retrieval is required. The method shown here uses a strong chelating agent, EDTA, and has been found efficient for the majority of antigens investigated. Solutions Required AP Developing solution: For 50 ml developing solution add in order: 50 ml Tris-HCI O.lM pH 9.2 (1:10 from a stock solution 1M). Levamisole 1mM (12 mg/50 ml) (Sigma L9756). 20 mg Naphtol As BI phosphate (Sigma N2250, the cheapo) (stock solution 40 mg/ml in NN-DM formam ide, anhydrous, 0.5 ml aliquots kept at _20DC) . 10 mg Fast Blue BB Diazonium (Sigma F3378) salt or Fast Red Diazonium salt Shake well Filter with a 45 11m filter. Keep away from direct light, use within 5 min. HRP Developing solution: For 50 ml developing solution add in order: Aminoethylcarbazole (20 mg tablets, Sigma A-6926, dissolved in 2.5 ml NN-DM

I I

Animal Cell and Tissue Culture

429

formamide) 50 ml acetate buffer pH 5.5 (52.5 ml of O.IM acetic acid solution + 196.5 ml of a O.IM Na acetate solution, bring to 500 ml) 25 ml H,O, 30%. Shake w~lJ~ Filter with a 4511m filter (optional). Keep away from direct light, use within 5 min.

Section Preparation 1. Cut sections at 411m, use a clean water bath with distilled water, and let the sections dry upright in order to facilitate adhesion between the section and the charged glass surface. 2. In order to enhance adhesion, the usual recommendation is to "bake" the slides (30 min at 60°C or overnightat 37°C.) 3. Deparaffinize the slides with two 5 min. incubations of clean xylene, folJowed by three washes with absolute ethanol. Then gradualJy bring to distilled water. 4. Place the sections in a radiotransparent slide holder (not metal; WVRlBaxter slide staining holder S7636. 5. Immerse slides and holder in ImM EDTA pH 7.5 (from a 100 mM stock) in a beaker. Cover with a piece of Saran wrap in which you made holes. Put in a microwave oven and bring to a boil at max power (8 min for 800 ml). Let boil for 15 min at a reduced power (Power 3) so that the liquid continue to simmer. Cool at RT for 30 min to one hour. Transfer to TBS. Alternatively, a pressure cooker or an autoclave can be used, with optimal results. Perform all stainings in a humid chamber. NOTE: The moist chamber (see above) must fulfill the following criteria: a) be absolutely flat, b) maintain moisture over 3 days without drying. c) be washable. A. Double indirect AP immunohistochemistry (e.g rabbit anti antigen X, goat anti- rabbit AP, rabbit anti-goat AP)

Procedure

o o o o

o

o

o

Wash twice in TBS 0.05M pH 7.5, to which 0.01% Tween 20 has been added (TBS-T). Briefly blot the slides without letting them dry and then apply 3% human or pig serum in PBS-BSA-NaN3 as a blocking agent. Incubate with the blocking for 10 min. If one of your antibody is biotin-conjugated, you need at this point to do endogenous biotin blocking. Blot the slides without washing and apply the primary antibody (100 ml), in a moist chamber, at RT for 1-18 h. Wash twice in TBS- T. 5 min / wash. Add the AP conjugated secondary antibody (50 to 100 Ill) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use. SBA Goat anti mouse AP or Goat anti Rabbit APcan be used 1:200 in TBS-BSA- NaN 3• Wash thrice in TBS- T.

430

Techniques in Life Sciences

o

Add the AP conjugated tertiary antibody (50 to 100 Ill) and incubate for 15 min.The tertiary antibody should be absorbed against human serum; if not add 1% human serum before use. SBA Rabbit anti Goat-AP or Goat anti Rabbit AP can be used 1:300 and 1:200 respectively in TBS-BSA NaN 3• o Add the AP conjugated tertiary antibody (50 to 100 Ill) and incubate for 15 min.The Wash thrice in TBS-T. o Add theAP conjugated tertiary antibody (50 to 100 Ill) and incubate for 15 min.The Add 50 ml of the developing solution (see below). Protect from direct light. DAfter 5 min, check the staining in your positive and negative controls. o Check the staining at 10-15 min interval. o When staining is complete (usually < 1 h.), wash thoroughly in tap water. o Preferably postfix in formalin for 4-5 h. before mounting in water soluble m 0 u n ting medium (glycerol gelatin).

B.

Indirect immunohistochemistry with avidin-biotin peroxidase(e.g rabbit anti antigen X, goat anti-rabbit biotin, avidin HRP)

Procedure o Wash twice in TBS-T. 5 min / wash. o Briefly blot the slides without letting them dry and then apply egg white in PBSBSA-NaN3 as a blocking agent (one egg white in 100 ml PBS + NaN3) Alternatively, use commercial blocking kits. Wash briefly once. o Apply 3% human or pig serum a s a blocking agent for 10 min Wash briefly. o Incubate with skim milk 5% in TBS-T for 10-30 min. This is the free avidin blocking step. Wash twice in TBS-T. o Blot the slides without washing and apply the primary antibody, in a moist chamber, at RT for 1-18 hr. o Wash twice in TBS-T. 5 min / wash. o Add the biotin-conjugated secondary antibody (50 to 100 ~I) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use. Most reagents are used at 1: 100 or 1:200 dilution. o Wash twice in TBS-T. 8bis- block endogenous peroxidase by incubating in 0.I%NaN3 and 0.3% HP2 for 30 min. Wash thrice. o Add the HRP-conjugated avidin (50 to 100 ~l, dilution 1:300 -500) and incubate for 20 min. Be careful not to dilute the avidin-HRP in biotin-containing or azide-containing medium. o Wash thrice in TBS-T. o Add 50 ml of the developing solution (see below). Protect from direct light. DAfter 5 min, check the staining in your positive and negative controls. o Check the staining at 10-15 min interval. o When staining is complete (usually < 1 hr), wash thoroughly in tap water. o Counterstain and mount in water soluble mounting medium (glycerol gelatin) .

•••

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Runnebaum B., Rabe T. (1994). Gynakologische Endrokrinologie und Fortilanzungsmediziz. Springer verlag., New York. Sambrook, J., Russell, D.W. and Maniatis, T.E. (2001). Molecular Cloning. : A laboratory manual. III Edn.; Cold Spring Harbor, New York, USA. Searcy R. L. (1974). Diagnostic Biochemistry, McGraw Hill, New York, Searcy D. G and Macinnis, A. J. (1970a). Determination of DNA by the Burton diphenylamine technique. In Experiments and Techniques in Parasitology (Eds. Macinnis, A.J. and Voge, M.)., w.H. Freeman and Co. San Fransisco. USA. Searcy D. G and Macinnis, A. J. (1970 b). Determination of RNA by the Dische Orcinol technique. In Experiments and Techniques in, Parasitology (Eds. Macinnis, AJ. and Voge, M.)., W.H. Freeman and Co. San Fransisco. USA. Seifter S., Dayton S., Novic B. and Muntwyler E. (1950). Estimation of glycogen with anthrone reagent. Arch. Biochem. 25:191-200. Seligson, D. (Ed.) (1963) Standard Methods of Clinical Chemistry. Academic Press, New York. Seth J., Rutherford E J., McKenzie I. (1975). Solid-Phase radioimmunoassay of thyroxine in untreated serum. Clin. Chem 21: 1406 - 1413. Shapiro, H.M. (1988) Practical Flow Cytometry, 2nd Edn. ,Alan R. Liss. Inc. , New York. Shchoneshofer M., Wagner G G, (1977). Sex differences in corticosteroid in man, J. Clin. Endocrinol. Metabol. 45: 814- 817. Shimozawa K., Saisho S. (1984). A neonatal mass screening program for congenital adrenal hyperplasia in Japan, Acta Endocrinol. 38: 111-116. Singh, A. (2002). Fundamental Techniques in Immunology and Serology. Internat. Book Distributing Co., Lucknow. Sippel W. G., Dorr H. G., Bidlingmaier E, Knorr D. (1980). Plasma levels of 17hydroxyprogesterone, Cortisol, and cortisone during infancy and childhood, Pediatr. Res. 14: 39- 46. Slater, R.J. (1985) Experiments in Molecular Biology. Humana Press. New Jersey. Snell, ED. and Snell, C.T. (1949) Colorimetric Methods of Aanalysis. Van Nstrang Co. Inc. New Jersey, Snyder, L.R. and Kirkland, J.J.(1980) Introduction to Modern Liquid Chromatography. 2nd edn. Wiley - Interscience, New York.

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Sobotka, U. and Stewart,T.P. (Eds). (1962). Advances in Clinical Chemistry. Academic Press, New York. Solomon D. H., Benotti J., Degrade L., (1972). A nomenclature for tests of thyroid hormones in serum: Report of a committee of the American Thyroid Association, J. Clin. Endocrinol. Metab. 34: 884-890. Southern, E. (1979). In: Methods of Enzymology (Ed. Wee, G) Academic Press, New York. Steele W. J., (1968) Localization of DNA complimentary to ribosomal ribonucleic acid and preribosomal ribonucleic acid in the nucleolus of rat liver. J. Bioi. Chem., 243: 33333341 Tabor, H. and Tabor, C.W. (1970). Methods in Enzymology. Academic Press New York. Talwar G .P. and Gupta S. K (1992) A Handbook of Practical Clinical Immunology. CBS Publishers and Distributors, New Delhi. Tembhare D.B. (1984) A Text book of Insect Morphology, Physiology and Endocrinology, S. Chand & Co. New Delhi. Tembhare D.B. (1998) Modern Entomology, Himalaya Publ. House, Mumbai, India. Tembhare, D.B. & Thakare, V.K. (1975) The histological and histochemical studies on the ovary in relation to vitellogenesis in the dragonfly, Orthetrum chrysis (Selys). Z. milkroskanat, Forsch, Leipzig, 89: 108-127. Tembhare, D.B, Thakare, Y.K,. & Deshmukh, KB. (1975). Free amino acids in the haemolymph of the last instar nymph of the dragonfly, Orthetrum chrysis (Selys). Experientia, 31: 1472-1473. Tembhare, D.B, Thakare, Y.K &. (1975) Histochemistry of the neurosecretory cells of the pars intercerebralis of the dragonfly, Orthetrum chrysis (Selys). Odonatologica, 4: 225235. Tembhare, D.B., Thakare, Y.K, & Deshmukh, KB. (1975) Free amino acids in the haemolymph of the last instar nymph of the dragonfly, Orthetrum chrysis (Selys). Experientia, 31: 1472-1473. Tembhare, D.B. & Thakare, V.K (1977) Neurosecretory system of the ventral ganglia in the dragonfly, Orthetrum chrysis (Selys). Cell Tiss. Res., 177 : 269-280. Tembhare, D.B. & Thakare, V.K (1977). Light microscopic structure and steroid histochemistry of the ovary in relation to steroidogenesis in the dragonfly, Orthetrum chrysis (Selys). Zoologica Poloniae, 26 : 557-572.

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Tembhare, D.B. (1979). Hormonal regulation of osmotic and ionic balance in the haemolymph of the larvae of dragonfly, Aeschna cyanea (MOHer). Arch. Internal. Physiol. Biochim, 87 : 557-563. Tembhare, D.B. (1980). An electron microscopic study of the neurosecretory pars intercerebraliscorpus cardiacum system in the larvae of the dragonfly, Aeschna cyanea (MilHer). Z. mikrosk-anat, Forsch, Leipzig, 94 (1980): 60-72. Tembhare, D.B., Thakare Y.K., Khan, M.W. and Pendharkar V. (1980) Chemical composition of haemolymph in the larvae of the dragonfly. Orthetrum chrysis (Selys). Odonotologica, 9: 321-324. Tembhare, D.B. & Andrew, R.J., (1992) Surface ultrastructure of the egg chorion in the dragonfly, lctinogomphus rapax (Rambur). Int. 1. Insect Morphol. EmbryoL 21 : 347-350. Tembhare, D.B. & Andrew, RJ. (1993) Functional anatomy of the secondary copulatory apparatus of the male dragonfly, Tramea virginia. 1. Morphol( USA). 218 : 99-106. Tembhare, D. B. & Barsagade, D. D. (2003) Effect of microbial infection on the posterior silkgland in the tropical tasar silkworm, Antheraea mylitta (Drury) (Lepidoptera: Satumiidae) Entomon, 28 (3) : 231 - 235. Tembhare, D.B. & Indurkar, U.S. (2005) Immunohistochemical localization of vertebrate gastro-intestinal hormone-like substances and their role in the midgut digestive enzyme activity in Cybister tripunctatus 01. (Coleoptera: Dytiscidae) Entomon 30 (4) : 279 288. Tietz N. (Ed) (1976).Analytical procedures and instrumentation, Fundamentals of Clinical Chemistry W. B. Saunders and Co. Philadelphia. Tietz N. W. (1983). Clinical Guide to Laboratory Tests, W. B. Saunders, Philadelphia Tietz N. W. (Ed.) (1986). Textbook of Clinical Chemistry .W. B. Saunders, Philadelphia. Thomeycroft I. H., Mishell D. R, Stone S. C., Kharma K. M., Nakamura R M. (1971). The relation of serum 17-hydroxyprogesterone and estradiol-17p during the human menstrual cycle. Am. 1. Obstet. Gynecol.Ill: 947- 951. Tortora GJ., and Grabowski, S.R (2003). Principles of Anatomy and Physiology., 10th Edn. John Wiley & Sons, Inc.New York, USA. Trevan M.D., Boffey, S., Goulding, KH., and Stanbury, P. (1988). Biotechnology - The Principles, Tata McGraw Hill" New Delhi, India. Upadhyay, A.; Upadhyay, K. and Nath, N. (2007). Biophysical Chemistry. Himalaya Publ. House, Mumbai., India.

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Varely H. (1980). Practical Clinical Biochemistry, Vol. 1: (5th ed.), William Heinemann Medical Books Ltd. London Van Vunakis,

D and Langone, 11 (1980). Methods in Enzymology.

Academic Press, New York.

Vaishnav Y. P. (1974). Test Book of Clinical Pathology, 1st Edition, Mohini Prakashan, Baroda. Walker, J.M. (1984) In : Methods in molecular biology. Humana Press. New Jersey. Vol. 1, Warnick G. R, Nguyen. T. and Alberts A. A. (1985). Comparision of improved precipitation methods for quantification of high density lipoprotein cholesterol. Clin. Chem. 31: 217219. Wehhmann R E., (1985). Suppression of thyrotropin in the low-thyroxine state of severe nonthyroidal illness. N. Eng. 1. Med., 312: 546- 52. Whelan, w.I. (1975). Biochemistry of Carbohydrates. Butterworth and University Park Press. London. Whistler, RL. (1962 - 71) Methods in Carbohydrate Chemistry. Vol. 1-6. Academic Press, New York. Wilkinson J. H. (1976). The Principle and Practice on Diagnostic Enzymology .Year book medical publishers. Willium J and Marshall Mosby (1995) Clinical Chemistry, 3rd edition, London. Wilson K. and Goulding (1986). A Biologist's Guide to Principles and Techniques of Practical Biochemistry. ELBS Publication, New Delhi. Wilson, K. and Walker, John (2005). Principles and Techniques of Biochemistry and Molecular Biology. Cambridge Univ. Press, London. Witherspoon, L. R, Shuler S. E., Garcia M. M., (1979). Effect of contaminant radioactivity on results of 1251-radiolegand assay. Clin. Chem. 25: 1975-1977. Wim, Gaastra (1984). In: Methods in molecular biology. Vol.l.Proteins (Ed. J.M. Walker) Humana Press New Jersey. Yamaguchi, I, Matsumura, F. and Kadous, A. A. (1979). Inhibition of synaptic ATPase by heptachlorepoxide in rat brain. Pest. Biochem. Physiol. 11 : 285 - 293. Young, D.S. (1976). Fundamentals of Clinical Chemistry, W. B. Saunders Co. Philadelphia Young D. S. (1990). Effect of drugs on Clinical Laboratory Test (3rd Ed.) AACC Press, Washington D. C. Zugibe, Fr (1970) Diagnostic Histochemistry. C.Y. Mosby, St. Louis.

• ••

APPENDIX 1.

SOLUTIONS

Molar (Molecular) solutions A molecular solution (M ) contains the molecular weight in grams of the substance made up to 1 liter with distilled water. Molecular weight expressed in grams is called the grammolecular weight or mole. A milimole is 111000 of a mole. In other words, one molar solution of a substance contains one mole or one gram molecular weight of a substance in one litter of solution. Eg.: 1M NaOH solution contains 40 g of NaOH in one litre solution. Molarity (M)

The number of moles of solute per litre of solution.

Molarity (m)

The number of moles of solute per kg of solvent. No. of moles NoofL Atomic weight of an element. i.e. - Atomic weight of Na = 23.

m=

Atomic weight Molecular weight

The sum of atomic weght of all atoms of a molecule.i.e.molecular weight of H 2S04 =98 { 2 X 1(hydrogen) =2 + 1 X 32 (Sulphur) = 32 = 4X16 (oxygen) = 64}.

Equivalent weight

A number of grams of the substance required to react with , replace or furnish one mole of ~O+ or OH- . Le., Equivalent weight of H 2S04 =49.

Normal solutions

A normal solution (N) contains 1 gram-molecular weight of dissolved substance divided by the hydrogen equivalent of the substance i.e., 1 gram equivalent, per lit. of solution. No. of g equivalent N=------1 Lit. Le., IN ~S04 solution contains 49 g of H 2S04in one lit. solution.

Techniques in Life Sciences

444 Dilutions for 1N Solutions Reagent

Molecular Weight

g / liter

mllliter

Acetic acid (CH3COOH)

60.05

1050

57.20

Ammonium hydroxide (NHPH)

17.03

251.40

67.70

Formic acid (HCOOH)

46.03

II 94

38.40

Hydrochloric acid (Hel)

36.46

451.60

80.40

Nitric acid (HN0 3)

63.02

989.4

63.60

Sulphuric acid (H 2SO 4)

98.075

1762

27.80

Calculated from most commonly available form (purity percentage) of the reagents.

Percentage solutions Percentage of solutions should be expresses in weight or volume w/v v/v

=weight in grams in a 100 ml volume of solution =volume in milliliters in a 100 ml volume of solution % Solution

Weight of Solute

= Weight of SolutIOn .

x 100

The correct v/v dilution is for example, a 1% solution of acetic acid is 1 ml of glacial acetic acid in 99 ml of distiIled water.

Hydrogen Ion Concentration (pH) pH can be defined as the negative logarithum (base 10) of the hydrogen or hydronium ion concentration in moles per litre. When pH is above 7 the solution is basic and when it is below 7 the solution is acidic. The pH of a neutral solution can, therefore, be written as, + ·7 pH = - loglO (H ) = loglO [1 x 10 ] =7. .

ButTer Solutions A solution containing both a weak acid and its conjugate weak base whose pH changes only slightly on addition on an acid or alkali.

Mixtures Acid alcohol: 1 m1 of HCl concentrated into 100 ml of 70% ethyl alcohol. Alkaline alcohol: 1 ml of ammonia, concentrated (or saturated aqueous sodium or lithium bicarbonate) into 100 ml of 70% ethyl alcohol. Carbol Xylol: Mixture of 1 part of phenol (carbolic acid) and 3 parts of xylene.

445

Appendix 2.

BUFFER SOLUTIONS

2.1

Common Buffer Ingredients Molecular weight

Buffer ingredients Acetic acid, CH3COOH

60.05

Borax, NaB P7.IOHp (Sodium tetraborate) Boric acid, B(OH)3

381.43 61.84

Citric acid (anhydrous), C3H4 (OH)(COOH) Citric acid crystals, C 3H4 (OH)(COOH)3.HP

192.12 210.14

Formic acid, HCOOH

46.03

Hydrochloric acid, HCl

36.46

Maleic acid, HOOCCH-CHOOH

116.07

Potassium acid phosphate, KI\P04 Sodium acetate, CH3COONa

136.09

Sodium acetate, crystals, CI\COONa.3Hp

136.09

Sodium barbital, CgHII03N2Na

206.18

Sodium bicarbonate, NaHC03

84.02

82.04

Sodium carbonate, NaC03 Sodium citrate, crystals, C3HPH(COONa)3' 5Hp

106.00 348.17

Sodium citrate, crystals, C3HPH(COONa)3' 5YzHp Sodium citrate,granular, C 3HPH(COONa)3' 2Hp

294.12

Sodium hydroxide, NaOH

40. 00

357.18

Sodium phosphate, monobasic, NaHl04. Hp

138.01

Sodium phosphate, dibasic, NaHP0 4. 7Hp Sodium phosphate, dibasic, NaHP04. Anhydrous

268. 14

Sulphuric acid,

~SO 4

98. 08

Tris (hydroxy methyl) aminomethane, C4H ll N0 3 2.2

141. 98 121. 14

Types of Buffers

2.2.1 O.2M Acetate Buffer (Gomori)

Stock solutions Solution. A: Acetic acid-

12.0 ml acetic acid in 988 ml distilled water.

Solution B: Sodium acetate

27.2 g sodium acetate in 1000 ml distilled water

Add a few crystals of camphor to both solutions. For different pH buffer solutions mix as follows:

Techniques in Life Sciences

446

IiI

Solution A (m)) 87.0 80.0 73.0 62.0 51.0 40.0 30.0 21.0 14.5 11.0

3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6

Solution B (m)) 13.0 20.0 27.0 38.0 49.0 60.0 70.0 79.0 85.5 89.0

2.2.2. Acetate - Acetic Acid Buffer (Walpole)

Stock Solutions

MI5 Acetic acid -

12.0 ml (99% assay) made up to 1000 ml with distilled water.

MI5 Sodium acetate -

27.2 g in 1000 ml distilled water.

pH

2.70 2.80 2.91 3.08 3.14 3.20 3.31 3.41 3.59 3.72 3.90 4.04 4.16 4.27 4.36 4.45 4.53 4.62 4.71 4.80 4.90

MJS Acetic Acid (ml) 200.0 199.0 198.0 196.0 195.0 194.0 192.0 190.0 185.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 090.0 080.0 070.0

MJS Sodium Acetate (mt) 000.0 001.0 002.0 004.0 005.0 006.0 008.0 010.0 015.0 020.0 030.0 040.0 050.0 060.0 070.0 080.0 090.0 100.0 110.0 120.0 130.0

447

Appendix

2.2.3 0.05 M Barbital Buffer Stock Solution Sodium barbital-

1.03 9 in 50 ml distilled water

Add following amount of 0.1 N Hel to 50 ml sodium barbital solution to obtain desirable pH buffer solution:

pH

O.lN HCI (ml)

pH

O.lN HCI (ml)

8.7 8.5

5.0 7.5

7.65 7.45

26.0 31.0

8.3

11.0

7.3

36.0

8.1

15.0

7.15

41.0

7.9

19.0

6.9

43.5

2.2.4. 0.2 M Phosphate Buffer Stock Solutions Monobasic sodium phosphate (MSP)27.6 g monobasic sodium phosphate in 1000 distilled water.

Dibasic sodium phosphate (DSP)53.6 g dibasic sodium phosphate in 1000 distilled water. For desirable pH buffer solution, mix both the solutions as given below:

pH

MSP (ml)

DSP (ml)

5.9

90.0

10.0

6.1

85.0

15.0

6.3

77.0

23.0

6.5

68.0

32.0

6.7

57.0

43.0

6.9

45.0

55.0

7.1

33.0

67.0

7.3

23.0

77.0

7.4

19.0

81.0

7.5

16.0

84.0

7.7

10.0

90.0

Techniques in Life Sciences

448 2.2.5 O.2M Tris Buffer Stock solutions

0.2 M tris (hydroxymethyl) aminomethane- 24.228 g tris aminomethane in 1000 ml

distilled water 0.1 N Hel (38% assay)

8.08 ml 0.1 N Hel in 991.92 distilled water.

Add O.IN Hel to 25 ml of 0.2 M tris as given below and dilute to 100 ml to obtain desirable pH:

pH

HCI (ml)

pH

HCI (ml)

7.19

45.0

8.14

25.0

7.36

42.5

8.23

22.5

7.54

40.0

8.32

20.0

7.66

37.5

8.41

17.5

7.77

35.0

8.51

15.0

7.87

32.5

8.62

12.5

7.96

30.0

8.74

10.0

8.05

27.5

8.92

07.5

2.2.6 Tris Maleate Buffer Stock solution

Maleic acid

29 g

Tris (hydroxymethyl) aminomethane

30.3 g

Distilled water

500ml

~

Add 2 g charcoal, shake, stand for 10 min and filter. Add 1N NaOH (4%) to 40 ml of stock solution as below and dilute to 100 ml for desirable pH:

pH

NaOH (ml)

pH

NaOH (ml)

5.8

9.0

7.0

19.0

6.0

10.5

7.2

20.0

6.2

13.0

7.6

22.5

6.4

15.0

7.8

24.2

6.6

16.5

8.0

26.0

6.8

18.0

8.2

29.0

4,

I

-

449

Appendix

2.2.7. Standard Buffer (Mc I1vaine) Stock solutions 0.1 M Citric acid anhydrous- 19.212 g citric acid anhydrous in 1000 ml distilled water. 0.2M disodium phosphate 28.396 g (.7H20, 53.628 g) in 1000 ml distilled water. (anhydrous) Mix both the solutions as below for desirable pH buffer solution:

,

t

! t

f

..

pH 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0

7.2

Citric acid (ml) 19.6 17.76 17.82 16.83 15.89 15.06 14.3 13.56 12.9 12.29 11.72 11.18 10.65 10.14 9.7 9.28 8.85 8.4 7.91 7.37 6.78 6.15 5.45 4.55 3.53 2.61

Disodium phosphate (ml) 0.4 1.24 2.18 3.17 4.11 4.94 5.7 6.44 7.1 7.71 8.28 8.82 9.35 9.86 10.3 10.72 11.15 11.6 12.09 12.63 13.22 13.85 14.55 15.45 16.47 17.39

7.4

1.83

18.17

7.6

1.27

18.73

7.8

0.85

19.15

8.0

0.55

19.45

Techniques in Life Sciences

450 2.2.8 Boric Acid - Borax Buffer Stock solutions Ml5 boric acid-

12.368g boric acid in 1000 ml distilled acid.

Ml20 borax-

19.071g borax in 1000 ml distilled water.

Mix both the solutions as below for desirable pH:

IiI

Ml5Boric acid (ml)

Ml20 Borax (ml)

7.4

90.0

10.0

7.6

85.0

15.0

7.8

80.0

20.0

8.0

70.0

30.0

8.2

65.0

35.0

8.4

55.0

45.0

8.7

40.0

60.0

9.0

20.0

80.0

2.2.9 Cacodylate Buffer (0.2M)

Stock solutions

• I

Sodium cacodylate [Na (CH3)2AS0 2.3HP] - 4.28% aqueous 0.2 M HCl [Concentrated HCl (36-38% purity)]

-1 ml in 60.3 ml distilled water

Desired pH cacodylate buffer can be obtained by adjusting 0.2 M HCl added to 50 ml of sodium cacodylate solution and diluted to a volume of 200 ml as given below:

pH

0.2M HCI (ml)

7.4

2.7

7.2

4.2

7.0

6.3

6.8

9.3

6.6

13.3

6.4

18.3

2.2.10. 0.2M Citrate Buffer

Solution A: Citric acid

- 4.2 g citric acid in 100 ml distilled water.

Solution B: Sodium citrate

- 5.9 g sodium citrate in 100 ml distilled water.

A few cryst~ls of camphor are to be added to both the solutions. For desirable pH mix both the solusions as given below:

451

Appendix

PI

Solution A (ml)

3.4 3.6 3.8 4.0 4.2 4.5 4.8 5.0 5.3 5.5

80 76 70 65 61 55 46 40 35 30

Solution B (ml)

20 24 30 35 39 45 54 60 65 70

3. INDICATORS

.,,

t

Indicator Thymol blue (acidic) Topfer's reagent Bromophenol blue Methyl orange Congo red Bromocresol green Methyl red Chlorophenol red Bromocresol purple Litmus Bromothymol blue Neutral red Phenol red Cresol red Thymol blue (alkaline) Phenolphthalein Thymolphthalein Alizarin yellow -R Tropaeolin 0 Other indicators: Methyl violet

pH range 1.2 - 2.8 2.9 - 4.2 3.0 - 4.6 3.0 - 4.4 3.0 - 4.5 3.8-5.4 4.4- 6.2 5.0- 6.6 5.2 - 6.8 4.5 - 8.3 6.0 - 7.6 6.8 - 8.0 6.8 - 8.4 7.2 - 8.8 8.0- 9.6 8.3 - 10.0 9.3 - 10.5 10.1 -12.1 11.1 - 12.7

Colour change Red to yellow Red to yellow Yellow to blue Red to yellow Blue to red Yellow to green Red to yellow. Yellow to red Yellow to purple Red to blue Yellow to blue Yellow to red Yellow to red Yellow to red Yellow to blue Colorless to red colorless to blue col.less to yellow Yellow to orange

0.5 - 1.5

yellow to blue

Methyl yellow

2.9-4.0

Red to yellow

Paranitrophenol

6.2 - 7.5

Colorless to yellow

Solution 0.1 g; 4.3 ml 0.05N NaOH to 250ml with DW 0.5 g in 100 m195% alcohol. 0.1 g; 3 ml 0.05 N NaOH to 250 ml with DW 0.1 gin 100 ml DW 0.5 g; 90 ml DW; 10 ml alcohol. O.lg; 2.9 ml 0.5 N NaOH, to 250 ml with DW 0.05 g in 100 ml 50% alcohol. O.lg; 4.8 ml 0.05N NaOH to 250 ml with DW. O.lg; 3.7 ml 0.05N NaOH to 250 ml with DW O.lg; 3.2 ml 0.05N NaOH to 250 ml with DW 0.1 % in 80% ethanol. O.lg; 5.7 ml 0.05N NaOH to 250 ml with DW O.lg; 5.3 ml 0.05N NaOH to 250 ml with DW As above. 0.1 to I% in 50% alcohol. 0.1 % in alcohol. 0.1% inDW 0.1% in DW

452

4.

5.

Techniques in Life Sciences

PROTEIN MARKERS USED IN SDS GEL ELECTROPHORESIS Protein markers a- Lactalbumin a-Lactoglobulin Soya Trypsin inhibitor Trypsinogen, PMSF treated Carbinic anhydrase, frombovine erythrocytes Pepsin Egg albumin Pyruvate kinase, from chicken muscle Bovine albumin G-6-P kinase from rabbit muscle Phosphorylase - b from rabbit muscle Galactosidase from E. coli a-2 macroglobulin from human plasma Urease from jack bean SIUNITS Prefixes of SI Units These are never used alone but always applied as units, for example, 11m or Ilg or Ill. Symbol Prefix Notation Meaning 18 exaE 10 one trillion 15 P petaone biljard 10 teraT one billion 10 12 9 gigaG one miljard 10 megaM one million 106 3 k kiloOne thousand 10 da deca 10 Ten 10- 1 one tenth d deci 10- 2 centic one hundredth 3 10millim one thousandth, 6 10microone millionth 11 10-9 nanon one miljard 12 10p picoone billionth 10- 15 f femtoone biljardth 10- 18 a attoone trillionth

Molecular weight 14,200 18,400 20,100 24,000 29,000 34,000 45,000 58,000 66,000 84,000 97,000 116,000 180,000 272,000

the prefixes to symbols of various

Multiples and submultiples 1 000,000, 000, 000, 000,000. 1, 000, 000, 000, 000, 000. 1,000, 000,000,000. 1, 000, 000, 000. 1,000,000. 1,000. 10 0.1 0,01. 0,001 0,000,001 0,000,000,001 0,000,000,000,001 0,000,000,000,000,001 0,000,000, 000, 000, 000, 001

453

Appendix

6

SIUNITS

6.1

Units of Length 111m= 1x1o-6 m =1x 1()3nm =1x 1()4 A lA = O.1nm = 1 x 104 !Jm == 1 x 10 -10m 1nm = lOA = 1 x 10-3 !Jm = 1 x 10-9 m Units of Volume 1 litre (L) = 1 dm3 = 1O-3m3 1 mililitre (ml) = 1 cm3 = 1O-6m3

6.2

1 microlitre (U)

6.3

= 1 mm3 = 10-9m3

Units of Weight

6.4 ..

106 lQJ

= = = = = = = =

Megagram (Mg) Kilogram (Kg) Gram (g) Milligram (mg) Microgram(!Jg) Nanogram (ng) Picogram (pg) Femtogram (fg)

10-3 10-6 10-9 10- 12 10- 15

= = = = = = = =

1000 kilogram 1000 gram 1000 miligram 1000 microgram 1000 nanogram 1000 picogram 1000 femtogram 1000 attogram

Unit Conversions Temperature Conversions 320f 580f

=OOC = 10°C

80°F 86°F

=27°C =30°C

Concentration Conversions 1 part per million (ppm) Number of moles

= 1 mg

1 kg Weight in g

=

Img

1 Lit. of H20

=-1 Lit. - -of-solvent ---

1 milimolar solution

1 mole of solute

=1M - - - - - - 1 Lit. of solvent

.. . 1 mlhmolar solutIOn

0.001 mole of solute

=mM =-1 -----Lit. of solvent

1 micormolar solution

1 x 10-6 mole of solute

= ~M =O.OOlmM = ----:--.,-----1 Lit. of solvent

Techniques in Life Sciences

454

Weight Conversion

1g

1

= -453

lb

I

7.

I mg

= 1000

I Jlg

=

g

1 1000mg

= I X 10-6 g

COMMON GREEK ALPHABETS Symbol

Name

A

a

alpha

B

p

beta

r

'Y

gamma

~

a

delta

E

E

epsilon

Z

S

zeta