الجمعة، 30 يوليو 2021
الأربعاء، 21 يوليو 2021
Basic laboratory procedures in clinical bacteriology
Basic laboratory procedures in clinical bacteriology
Contents
Preface viii
Introduction 1
Quality assurance in bacteriology 2
Introduction 2
Definitions 2
Internal quality control 6
External quality assessment 16
PART I : Bacteriological investigations 19
Blood 20
Introduction 20
When and where bacteraemia may occur 20
Blood collection 20
Blood-culture media 22
Processing of blood cultures 23
Cerebrospinal fluid 25
Introduction 25
Collection and transportation of specimens 25
Macroscopic inspection 26
Microscopic examination 26
Preliminary identification 28
Susceptibility testing 29
Urine 30
Introduction 30
Specimen collection 30
Culture and interpretation 32
Interpretation of quantitative urine culture results 34
Identification 35
Susceptibility tests 36
Stool 37
Introduction 37
Etiological agents and clinical features 37
Appropriate use of laboratory resources 39
Collection and transport of stool specimens 40
Visual examination of stool specimens 41
Enrichment and inoculation of stool specimens 41
Media for enteric pathogens 42
Primary isolation 42
Preliminary identification of isolates 44
Final microbiological identification 50
Serological identification 54
Upper respiratory tract infections 60
Introduction 60
Normal flora of the pharynx 60
Bacterial agents of pharyngitis 61
Collection and dispatch of specimens 62
Direct microscopy 62
Culture and identification 63
Susceptibility testing 65
Lower respiratory tract infections 66
Introduction 66
The most common infections 66
Collection of sputum specimens 68
Processing of sputum in the laboratory (for
non-tuberculous infections) 68
Culture for Mycobacterium tuberculosis 72
Interpretation of cultures for M. tuberculosis 74
General note on safety 74
Sexually transmitted diseases 76
Introduction 76
Urethritis in men 77
Genital specimens from women 79
Specimens from genital ulcers 82
Purulent exudates, wounds and abscesses 86
Introduction 86
Commonly encountered clinical conditions and the
most frequent etiological agents 86
Collection and transportation of specimens 89
Macroscopic evaluation 90
Microscopic examination 91
Culture 92
Identification 93
Susceptibility testing 97
Anaerobic bacteriology 98
Introduction 98
Description of bacteria in relation to oxygen requirement 98
Bacteriology 98
Antimicrobial susceptibility testing 103
Introduction 103
General principles of antimicrobial susceptibility testing 103
Clinical definition of terms “resistant” and “susceptible”:
the three category system 104
Indications for routine susceptibility tests 106
Choice of drugs for routine susceptibility tests in the
clinical laboratory 107
The modified Kirby–Bauer method 109
Direct versus indirect susceptibility tests 117
Technical factors influencing the size of the zone in the
disc-diffusion method 118
Quality control 120
Serological tests 122
Introduction 122
Quality control measures 122
Serological reactions 125
Serological tests for syphilis 126
Febrile agglutinins tests 133
Antistreptolysin O test 135
Bacterial antigen tests 137
PART II: Essential media and reagents 141
Introduction 142
Pathogens, media and diagnostic reagents 143
Blood 144
Cerebrospinal fluid 144
Urine 145
Stool 146
Upper respiratory tract 147
Lower respiratory tract 148
Urogenital specimens for exclusion of sexually transmitted
diseases 149
Pus and exudates 149
List of recommended media and diagnostic reagents for the intermediate microbiological laboratory 150
Selected further reading 154
Index 155
Bacteriology of Humans
Bacteriology of Humans
An Ecological Perspective
Michael Wilson
University College London
CONTENTS
Preface, ix
Abbreviations of genera, xi
1 THE HUMAN–MICROBE SYMBIOSIS
1.1 Overview of the nature and distribution of the microbial communities inhabiting humans
1.1.1 Difficulties encountered in determining the composition of a microbial community, 2
1.1.2 Structural aspects of microbial communities, 5
1.1.2.1 Microcolonies, 5
1.1.2.2 Intracellular colonization, 5
1.1.2.3 Biofilms, 6
1.1.3 Communication in microbial communities, 8
1.2 Environmental determinants that affect the distribution and composition of microbial communities
1.2.1 Nutritional determinants, 14
1.2.2 Physicochemical determinants, 18
1.2.3 Mechanical determinants, 22
1.2.4 Biological determinants, 23
1.3 Host characteristics that affect the indigenous microbiota
1.3.1 Age, 28
1.3.2 Host genotype, 29
1.3.3 Gender, 29
1.4 Techniques used to characterize the microbial communities inhabiting humans
1.4.1 Microscopy, 31
1.4.2 Culture-dependent approaches, 33
1.4.3 Culture-independent, molecular approaches, 35
1.4.4 Functional analysis of microbial communities, 37
1.5 The epithelium – site of host–microbe interactions
1.5.1 Structure of epithelia, 38
1.5.2 The epithelium as an excluder of microbes, 41
1.5.3 Mucus and mucins, 41
1.5.4 Innate and acquired immune responses at the mucosal surface, 46
1.6 Further reading
1.6.1 Books, 53
1.6.2 Reviews and papers, 53
2 THE INDIGENOUS MICROBIOTA OF THE SKIN
2.1 Anatomy and physiology of human skin, 56
2.2 Cutaneous antimicrobial defense systems, 56
2.2.1 Innate defense systems, 58
2.2.2 Acquired immune defense systems, 60
2.3 Environmental determinants operating at different skin regions, 61
2.4 The indigenous microbiota of the skin, 67
2.4.1 Members of the cutaneous microbiota, 67
2.4.1.1 Corynebacterium spp., 67
2.4.1.2 Propionibacterium spp., 70
2.4.1.3 Staphylococcusspp., 71
2.4.1.4 Micrococcusspp., 73
2.4.1.5 Malassezia spp., 75
2.4.1.6 Acinetobacterspp., 76
2.4.1.7 Brevibacterium spp., 78
2.4.1.8 Dermabacter hominis, 79
2.4.1.9 Methylobacterium spp., 79
2.4.2 Community composition at different sites,80
2.4.3 Culture-independent studies of the cutaneous microbiota, 86
2.4.4 Interactions among members of the cutaneous microbiota, 88
2.5 Overview of the cutaneous microbiota, 90
2.6 Sources of data used to compile figures , 92
2.7 Further reading, 92
2.7.1 Books, 92
2.7.2 Reviews and papers, 92
3 THE INDIGENOUS MICROBIOTA OF THE EYE
3.1 Anatomy and physiology of the eye, 95
3.2 Antimicrobial defense systems of the eye, 97
3.3 Environmental determinants on the conjunctival surface, 99
3.4 The indigenous microbiota of the eye, 103
3.4.1 Members of the ocular microbiota, 103
3.4.2 Composition of the ocular microbiota, 104
3.4.3 Interactions among members of the ocular microbiota, 107
3.5 Overview of the ocular microbiota, 111
3.6 Sources of data used to compile figures, 111
3.7 Further reading, 111
4 THE INDIGENOUS MICROBIOTA OF THE RESPIRATORY TRACT
4.1 Anatomy and physiology of the respiratory tract, 113
4.1.1 Nose, 113
4.1.2 Pharynx, 114
4.1.3 Larynx, 114
4.1.4 Trachea, 114
4.1.5 Bronchi and bronchioles, 114
4.1.6 Alveolus, 115
4.2 Antimicrobial defense systems of the respiratory tract, 117
4.2.1 Nasal cavity, 117
4.2.2 Other regions of the conducting portion, 119
4.2.3 Respiratory portion, 119
4.3 Environmental determinants within the respiratory tract, 119
4.3.1 Atmospheric composition, 119
4.3.2 pH, 120
4.3.3 Nutrients, 120
4.3.3.1 Composition of nasal fluid, ASL, and alveolar lining fluid, 120
4.3.3.2 Contribution of microbial residents of the respiratory tract to nutrient availability, 122
4.4 Indigenous microbiota of the respiratory tract, 123
4.4.1 Members of the respiratory microbiota,123
4.4.1.1 Streptococcusspp., 123
4.4.1.1.1 Strep. pyogenes, 124
4.4.1.1.2 Strep. pneumoniae, 126
4.4.1.1.3 Viridans group streptococci, 128
4.4.1.2 Neisseria spp., 128
4.4.1.2.1 N. meningitidis, 129
4.4.1.2.2 Other Neisseria spp.,131
4.4.1.3 Haemophilusspp., 131
4.4.1.3.1 H. influenzae, 132
4.4.1.3.2 Other Haemophilusspp.,134
4.4.1.4 Moraxella catarrhalis, 135
4.4.1.5 Staphylococci, 136
4.4.1.5.1 Staph. aureus, 136
4.4.1.5.2 CNS, 138
4.4.1.6 Mollicutes, 139
4.4.1.7 Kingella kingae, 140
4.4.2 Community composition at the various sites within the respiratory tract, 140
4.4.2.1 Nasal vestibule, 142
4.4.2.2 Nasal cavity, 143
4.4.2.3 Nasopharynx, 146
4.4.2.4 Oropharynx, 146
4.4.2.5 Lower respiratory tract, 151
4.4.3 Interactions among members of the respiratory microbiota, 152
4.5 Overview of the respiratory microbiota, 152
4.6 Sources of data used to compile figures, 155
4.7 Further reading, 157
5 THE INDIGENOUS MICROBIOTA OF THE URINARY SYSTEM OF FEMALES
5.1 Anatomy and physiology of the urinary system of females, 159
5.2 Antimicrobial defenses of the female urinary system, 160
5.3 Environmental determinants within the female urethra, 161
5.4 The indigenous microbiota of the female urethra, 162
5.4.1 Members of the urethral microbiota, 163
5.4.2 Community composition in the female urethra, 164
5.5 Overview of the microbiota of the urinary tract of females, 167
5.6 Sources of data used to compile figures, 168
5.7 Further reading, 168
6 THE INDIGENOUS MICROBIOTA OF THE REPRODUCTIVE SYSTEM OF FEMALES
6.1 Anatomy and physiology of the female reproductive system, 170
6.2 Antimicrobial defense systems of the female reproductive system, 172
6.2.1 Innate defense systems, 173
6.2.2 Acquired immune defense systems, 174
6.3 Environmental determinants at different regions of the reproductive system, 176
6.3.1 Vagina, 176
6.3.2 Cervix, 179
6.3.3 Vulva, 179
6.3.4 Contribution of the indigenous microbiota to nutrient supply within the reproductive system, 179
6.4 The indigenous microbiota of the female reproductive system, 181
6.4.1 Members of the microbiota, 181
6.4.1.1 Lactobacillusspp., 181
6.4.1.2 Gardnerella vaginalis, 184
6.4.1.3 Candida albicans, 184
6.4.1.4 Streptococcus agalactiae(Group B streptococcus), 186
6.4.1.5 Mycoplasma hominis, 186
6.4.1.6 Ureaplasma urealyticum, 186
6.4.1.7 Atopobium vaginae, 187
6.4.1.8 Mobiluncusspp., 187
6.4.2 Community composition at different sites within the female reproductive system, 187
6.4.2.1 Vagina, 187
6.4.2.1.1 Post-menarcheal/pre- menopausal females, 187
6.4.2.1.2 Pre-menarcheal girls,191
6.4.2.1.3 Post-menopausal women, 192
6.4.2.1.4 Vaginal microbiota during pregnancy, 193
6.4.2.2 Cervix, 193
6.4.2.2.1 Post-menarcheal/pre- menopausal females, 197
6.4.2.2.2 Cervical microbiota during pregnancy, 197
6.4.2.3 Vulva, 199
6.4.3 Interactions between organisms colonizing the female reproductive system, 200
6.5 Overview of the microbiota of the female reproductive system, 202
6.6 Sources of data used to compile figures, 202
6.7 Further reading, 204
6.7.1 Books, 204
6.7.2 Reviews and papers, 204
7 THE INDIGENOUS MICROBIOTA OF THE URINARY AND REPRODUCTIVE SYSTEMS OF MALES
7.1 Anatomy and physiology, 207
7.2 Antimicrobial defenses of the male urinary and reproductive systems, 207
7.3 Environmental determinants within the male urinary and reproductive systems
7.4 The indigenous microbiota of the male urinary and reproductive systems, 211
7.4.1 Members of the microbiota, 211
7.4.2 Microbiota of the male urethra, 212
7.4.3 Microbiota of the glans penis, 216
7.4.4 Microbiota of the prostate, 218
7.5 Overview of the microbiota of the male urinary and reproductive systems, 218
7.6 Sources of data used to compile figures, 219
7.7 Further reading, 220
8 THE INDIGENOUS MICROBIOTA OF THE ORAL CAVITY
8.1 Anatomy and physiology of the oral cavity, 222
8.2 Antimicrobial defense systems of the oral cavity,225
8.3 Environmental determinants at the various siteswithin the oral cavity, 228
8.3.1 Mechanical determinants, 228
8.3.2 Nutritional determinants, 228
8.3.3 Physicochemical determinants, 231
8.4 The indigenous microbiota of the oral cavity, 232
8.4.1 Members of the oral microbiota, 235
8.4.1.1 Oral streptococci and related Gram-positive cocci, 235
8.4.1.2 Gemella spp., 235
8.4.1.3 Actinomycesspp., 236
8.4.1.4 Rothia dentocariosa, 237
8.4.1.5 Veillonella spp., 237
8.4.1.6 Anaerobic and microaerophilic Gram-negative rods, 237
8.4.1.6.1 Fusobacterium spp., 237
8.4.1.6.2 Porphyromonasspp.,
8.4.1.6.3 Prevotella spp., 239
8.4.1.6.4 Spirochaetes, 239
8.4.1.6.5 Other anaerobic species, 239
8.4.1.7 Facultatively anaerobic Gram-negative bacilli, 240
8.4.1.8 Mycoplasma spp., 240
8.4.1.9 Megasphaera spp., 241
8.4.2 Community composition at different sites, 241
8.4.2.1 Supragingival plaque, 241
8.4.2.2 Gingival crevice, 253
8.4.2.3 Tongue, 256
8.4.2.4 Other mucosal surfaces, 257
8.5 Overview of the oral microbiota, 261
8.6 Sources of data used to compile figures, 263
8.7 Further reading, 264
8.7.1 Books, 264
8.7.2 Reviews and papers, 264
9 THE INDIGENOUS MICROBIOTA OF THE GASTROINTESTINAL TRACT
9.1 Anatomy and physiology of the gastrointestinal tract, 267
9.2 Antimicrobial defense systems of the gastrointestinal tract, 272
9.2.1 Innate defense systems, 272
9.2.2 Acquired immune defense system, 275
9.3 Environmental determinants within different regions of the gastrointestinal tract, 276
9.3.1 Esophagus, 276
9.3.2 Stomach, 277
9.3.3 Small intestine, 277
9.3.4 Large intestine, 278
9.4 The indigenous microbiota of the gastrointestinal tract, 280
9.4.1 Members of the intestinal microbiota, 282
9.4.1.1 Bacteroides, 282
9.4.1.2 Eubacterium, 283
9.4.1.3 Roseburia, 284
9.4.1.4 Clostridium, 284
9.4.1.5 Bifidobacterium, 284
9.4.1.6 Enterococcus, 285
9.4.1.7 Helicobacter pylori, 286
9.4.1.8 Enterobacteriaceae, 286
9.4.1.9 Ruminococcus, 287
9.4.1.10 Methanogenic organisms, 287
9.4.1.11 Desulfovibrio, 287
9.4.1.12 Acidaminococcus, 288
9.4.1.13 Faecalibacterium prausnitzii, 288
9.4.2 Community composition in different regions of the intestinal tract, 288
9.4.2.1 Esophagus, 288
9.4.2.2 Stomach, 289
9.4.2.3 Small intestine, 295
9.4.2.3.1 Duodenum, 295
9.4.2.3.2 Jejunum, 295
9.4.2.3.3 Ileum, 301
9.4.2.4 Large intestine, 302
9.4.2.4.1 Cecum, 304
9.4.2.4.2 Colon, 306
9.4.2.4.3 Rectum, 316
9.4.3 Microbial interactions in the gastrointestinal tract, 317
9.5 Overview of the indigenous microbiota of the gastrointestinal tract, 320
9.6 Sources of data used to compile figures, 320
9.7 Further reading, 322
9.7.1 Books, 322
9.7.2 Reviews and papers, 322
10 THE FUTURE, 327
10.1 Further reading, 329
Index, 331
WHO laboratory manual for the Examination and processing of human semen (Fifth Edition)
WHO laboratory manual for the Examination and processing of human semen
FIFTH EDITION
Chapter 1 Background 1
1.1 Introduction 1
1.2 The fi fth edition 1 1.3 Scope of the manual 3PART I. SEMEN ANALYSIS
Chapter 2 Standard procedures 7
2.1 Introduction 7
2.2 Sample collection 10
2.2.1 Preparation 10 2.2.2 Collection of semen for diagnostic or research purposes 11 2.2.3 Sterile collection of semen for assisted reproduction 11 2.2.4 Sterile collection of semen for microbiological analysis 11 2.2.5 Collection of semen at home 12 2.2.6 Collection of semen by condom 12 2.2.7 Safe handling of specimens 132.3 Initial macroscopic examination 13
2.3.1 Liquefaction 13 2.3.2 Semen viscosity 14 2.3.3 Appearance of the ejaculate 15 2.3.4 Semen volume 15 2.3.5 Semen pH 162.4 Initial microscopic investigation 17
2.4.1 Thorough mixing and representative sampling of semen 17 2.4.2 Making a wet preparation 18 2.4.3 Aggregation of spermatozoa 19 2.4.4 Agglutination of spermatozoa 19 2.4.5 Cellular elements other than spermatozoa 212.5 Sperm motility 21
2.5.1 Categories of sperm movement 22 2.5.2 Preparing and assessing a sample for motility 22 2.5.3 Worked examples 25 2.5.4 Lower reference limit 262.6 Sperm vitality 26
2.6.1 Vitality test using eosin–nigrosin 27 2.6.2 Vitality test using eosin alone 29 2.6.3 Vitality test using hypo-osmotic swelling 302.7 Sperm numbers 32
2.7.1 Types of counting chamber 34 2.7.2 The improved Neubauer haemocytometer 34 2.7.3 Using the haemocytometer grid 35 2.7.4 Care of the counting chamber 35 2.7.5 Fixative for diluting semen 36 2.7.6 Importance of counting suffi cient spermatozoa 362.8 Routine counting procedure 37
2.8.1 Determining the required dilution 38 2.8.2 Preparing the dilutions and loading the haemocytometer chambers 39 2.8.3 Assessing sperm numbers in the counting chambers 41 2.8.4 Calculation of the concentration of spermatozoa in semen 43 2.8.5 Worked examples 43 2.8.6 Lower reference limit for sperm concentration 44 2.8.7 Calculation of the total number of spermatozoa in the ejaculate 44 2.8.8 Lower reference limit for total sperm number 442.9 Low sperm numbers: cryptozoospermia and suspected azoospermia 45
2.10 When an accurate assessment of low sperm numbers is not required 45
2.10.1 Taking no further action 45 2.10.2 Examination of centrifuged samples to detect spermatozoa 45 2.10.3 Examination of non-centrifuged samples to detect motile spermatozoa 462.11 When an accurate assessment of low sperm numbers is required 48
2.11.1 Assessing low sperm numbers in the entire improved Neubauer chamber (phase-contrast microscopy) 48 2.11.2 Assessing low sperm numbers in large-volume disposable chambers (fl uorescence microscopy) 522.12 Counting of cells other than spermatozoa 55
2.12.1 Calculation of the concentration of round cells in semen 55 2.12.2 Sensitivity of the method 56 2.12.3 Worked examples 562.13 Sperm morphology 56
2.13.1 The concept of normal spermatozoa 57 2.13.2 Preparation of semen smears 582.14 Staining methods 62
2.14.1 Traditional fixation and sequential staining 62 2.14.2 Papanicolaou staining procedure for sperm morphology 63 2.14.3 Shorr staining procedure for sperm morphology 65 2.14.4 Rapid staining procedure for sperm morphology 662.15 Examining the stained preparation 67
2.15.1 Classification of normal sperm morphology 67 2.15.2 Classifi cation of abnormal sperm morphology 692.16 Morphology plates 70
2.17 Analysing a sperm morphology smear 99
2.17.1 Assessment of normal sperm morphology 99 2.17.2 Worked examples 100 2.17.3 Lower reference limit 100 2.17.4 Assessment of abnormal sperm morphology 101 2.17.5 Worked example 101 2.17.6 Assessment of specifi c sperm defects 1022.18 Assessment of leukocytes in semen 102
2.18.1 Staining cellular peroxidase using ortho-toluidine 1032.19 Assessment of immature germ cells in semen 107
2.20 Testing for antibody coating of spermatozoa 108 2.20.1 The mixed antiglobulin reaction test 109 2.20.2 The direct immunobead test 111 2.20.3 The indirect immunobead test 113Chapter 3 Optional procedures 115
3.1 Indices of multiple sperm defects 115
3.1.1 Calculation of indices of multiple morphological defects 115 3.1.2 Worked example 1163.2 Panleukocyte (CD45) immunocytochemical staining 117
3.2.1 Principle 117 3.2.2 Reagents 118 3.2.3 Procedure 1183.3 Interaction between spermatozoa and cervical mucus 122
3.3.1 In-vivo (postcoital) test 122 3.3.2 In-vitro tests 125 3.3.3 In-vitro simplifi ed slide test 1263.3.4 Capillary tube test 127
3.4 Biochemical assays for accessory sex organ function 130 3.4.1 Measurement of zinc in seminal plasma 130 3.4.2 Measurement of fructose in seminal plasma 132 3.4.3 Measurement of neutral -glucosidase in seminal plasma 1343.5 Computer-aided sperm analysis 136
3.5.1 Introduction 136 3.5.2 Use of CASA to assess sperm motility 137 3.5.3 Use of CASA to estimate sperm concentration 1403.5.4 Computer-aided sperm morphometric assessment 140
Chapter 4 Research procedures 142
4.1 Reactive oxygen species 142
4.1.1 Introduction 142 4.1.2 Measurement of reactive oxygen species generated by sperm suspensions 1434.2 Human sperm–oocyte interaction tests 146
4.3 Human zona pellucida binding tests 146
4.4 Assessment of the acrosome reaction 147
4.4.1 Procedure for the fluorescence assessment of acrosomal status 147 4.4.2 Induced acrosome reaction assay 150 4.5 Zona-free hamster oocyte penetration test 1524.5.1 Protocol 152
4.6 Assessment of sperm chromatin 157PART II. SPERM PREPARATION
Chapter 5 Sperm preparation techniques 161
5.1 Introduction 161
5.1.1 When spermatozoa may need to be separated from seminal plasma 161 5.1.2 Choice of method 161 5.1.3 Effi ciency of sperm separation from seminal plasma and infectious organisms 1625.2 General principles 162
5.3 Simple washing 163
5.3.1 Reagents 163 5.3.2 Procedure 1635.4 Direct swim-up 164
5.4.1 Reagents 164 5.4.2 Procedure 1645.5 Discontinuous density gradients 165
5.5.1 Reagents 165 5.5.2 Procedure 1665.6 Preparing HIV-infected semen samples 166
5.7 Preparing testicular and epididymal spermatozoa 167
5.7.1 Enzymatic method 167 5.7.2 Mechanical method 167 5.7.3 Processing sperm suspensions for intracytoplasmic sperm injection 1675.8 Preparing retrograde ejaculation samples 168
5.9 Preparing assisted ejaculation samples 168
Chapter 6 Cryopreservation of spermatozoa 169
6.1 Introduction 169
6.2 Semen cryopreservation protocols 172
6.2.1 Standard procedure 172 6.2.2 Modifi ed freezing protocols for oligozoospermia and surgically retrieved spermatozoa 175 6.2.3 Labelling of straws and records 176PART III. QUALITY ASSURANCE
Chapter 7 Quality assurance and quality control 179
7.1 Controlling for quality in the andrology laboratory 179
7.2 The nature of errors in semen analysis 179
7.3 Minimizing statistical sampling error 180
7.4 The quality assurance programme 182
7.5 Laboratory procedures manual 182
7.6 Internal quality control 182
7.6.1 Purchased QC samples 183 7.6.2 Laboratory-made QC samples 183 7.6.3 Stored samples (purchased or laboratory-made) 183 7.6.4 Fresh QC samples (laboratory-made) 1847.7 Statistical procedures for analysing and reporting within- and among-technician systematic errors 185
7.7.1 The Xbar chart 185 7.7.2 The S chart 1887.8 QC for percentages 189
7.9 Assessing Xbar and S charts 189
7.9.1 How to recognize out-of-control values 189 7.9.2 Causes of out-of-control values 190 7.9.3 Responses to out-of-control values 1917.10 Statistical procedures for analysing and reporting among-technician variability 191
7.10.1 Comparing results from two or more technicians 191 7.10.2 Monitoring monthly means 1947.11 External quality control and quality assurance 194
7.11.1 Assessment of EQC results 196 7.11.2 Responses to out-of-control results 1977.12 Frequency and priority of quality control 197
7.13 Training 198
7.13.1 Practical hints when experiencing difficulty assessing sperm concentration 198 7.13.2 Practical hints when experiencing diffi culty assessing sperm morphology 200 7.13.3 Practical hints when experiencing diffi culty assessing sperm motility 200 7.13.4 Practical hints when experiencing diffi culty assessing sperm vitality 202REFERENCES 205
APPENDICES
Appendix 1 Reference values and semen nomenclature 223
A1.1 Reference values 223 A1.2 Nomenclature 225Appendix 2 Equipment and safety 227
A2.1 Basic supplies needed in an andrology laboratory 227 A2.2 Potential biohazards in an andrology laboratory 230 A2.3 Safety procedures for laboratory personnel 230 A2.4 Safety procedures for laboratory equipment 232 A2.5 Safety precautions when handling liquid nitrogen 233Appendix 3 Microscopy 234
A3.1 Loading the sample 234 A3.2 Adjusting the oculars 236 A3.3 Focusing the image 236 A3.4 Focusing the oculars 236 A3.5 Focusing the light condenser 236 A3.6 Centring the condenser 237 A3.7 Adjusting the phase rings 237 A3.8 Fluorescence microscopy 237Appendix 4 Stock solutions 238
A4.1 Biggers, Whitten and Whittingham 238 A4.2 Dulbecco’s phosphate-buffered saline 238 A4.3 Earle’s medium 239 A4.4 Ham’s F-10 medium 239 A4.5 Hanks’ balanced salt solution 240 A4.6 Human tubal fl uid 240 A4.7 Krebs–Ringer medium 240 A4.8 Tris-buffered saline 241 A4.9 Tyrode’s solution 241 A4.10 Papanicolaou stain 241Appendix 5 Cervical mucus 245
A5.1 Introduction 245 A5.2 Collection and preservation of cervical mucus 246 A5.3 Evaluation of cervical mucus 247Appendix 6 Record forms for semen and cervical mucus analyses 251
A6.1 Template for a semen analysis recording form 251 A6.2 Template for a cervical mucus recording form 253Appendix 7 Sampling errors and quality control 254
A7.1 Errors in measurement of sperm concentration 254 A7.2 The importance of understanding sampling errors 256 A7.3 Errors in measurement of percentages 257 A7.4 Production of semen samples for quality control 260 A7.5 Preparation of a video-recording for internal quality control of analysis of sperm motility 261 A7.6 Preparation of diluted semen for internal quality control of determination of sperm concentration 265 A7.7 Preparation of slides for internal quality control of assessment of sperm morphology 268 A7.8 Calibration of equipment 269Appendix 8 National external quality control programmes for semen analysis 271
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