Manual basic techniques for a health laboratory

1. Introduction 1 1.1 Aim of the manual 1 1.2 Reagents and equipment 1 1.2.1 Reagents 1 1.2.2 Equipment 1 1.3 The responsibility of laboratory workers 2 1.4 Units of measurement 2 1.4.1 Quantities and units in the clinical laboratory 2 1.4.2 SI units and names for quantities 2 PART I 9 2. Setting up a peripheral health laboratory 11 2.1 Plan of a peripheral medical laboratory 11 2.1.1 A one-room laboratory 11 2.1.2 A two-room laboratory 12 2.2 Electricity 12 2.2.1 Sources of electricity 13 2.2.2 Setting up simple electrical equipment 15 2.2.3 What to do in case of failure of electrical equipment 17 2.3 Plumbing: simple procedures 20 2.3.1 Tools and materials 20 2.3.2 Taps 20 2.3.3 Sink traps 22 2.4 Water for laboratory use 23 2.4.1 Clean water 24 2.4.2 Distilled water 24 2.4.3 Demineralized water 27 2.4.4 Buffered water 29 2.5 Equipment 32 2.5.1 Essential laboratory instruments 32 2.5.2 Additional items 33 2.5.3 Equipment and supplies 33 2.5.4 Making glass equipment 33 2.5.5 Specimen containers 42 2.5.6 Storage, stocktaking and ordering supplies 45 2.6 Registration of specimens and preparation of monthly reports 46 2.6.1 Registration of specimens 46 iii iv Manual of basic techniques for a health laboratory 2.6.2 Preparation of monthly reports 47 3. General laboratory procedures 53 3.1 Use of a microscope 53 3.1.1 Components of a microscope 53 3.1.2 Setting up the microscope 58 3.1.3 Focusing the objective 61 3.1.4 Use of an ocular micrometer 63 3.1.5 Dark-field microscopy 64 3.1.6 Routine maintenance 64 3.2 Weighing: use of laboratory balances 66 3.2.1 Sensitivity of a balance 67 3.2.2 Open two-pan balance 67 3.2.3 Analytical balance 68 3.2.4 Dispensary balance 69 3.3 Centrifugation 69 3.3.1 Principle 69 3.3.2 Types of centrifuge 70 3.3.3 Instructions for use 71 3.4 Measurement and dispensing of liquids 73 3.4.1 Pipettes 73 3.4.2 Volumetric flasks 75 3.4.3 Burettes 77 3.4.4 Graduated conical glasses 77 3.5 Cleaning, disinfection and sterilization 77 3.5.1 Cleaning glassware and reusable syringes and needles 77 3.5.2 Cleaning non-disposable specimen containers 81 3.5.3 Cleaning and maintenance of other laboratory equipment 83 3.5.4 Disinfectants 83 3.5.5 Sterilization 85 3.6 Disposal of laboratory waste 90 3.6.1 Disposal of specimens and contaminated material 90 3.6.2 Incineration of disposable materials 90 3.6.3 Burial of disposable materials 91 3.7 Dispatch of specimens to a reference laboratory 91 3.7.1 Packing specimens for dispatch 91 3.7.2 Fixation and dispatch of biopsy specimens for histopathological examination 95 3.8 Safety in the laboratory 96 3.8.1 Precautions to prevent accidents 97 3.8.2 First aid in laboratory accidents 98 3.9 Quality assurance in the laboratory 101 3.9.1 Specimen collection 102 PART II 103 4. Parasitology 105 4.1 Introduction 105 4.2 Examination of stool specimens for parasites 107 Contents v 4.2.1 Collection of specimens 107 4.2.2 Visual examination 107 4.2.3 Microscopic examination 107 4.2.4 Dispatch of stools for detection of parasites 109 4.3 Intestinal protozoa 111 4.3.1 Identification of motile forms (trophozoites) 111 4.3.2 Identification of cysts 118 4.4 Intestinal helminths 125 4.4.1 Identification of eggs 126 4.4.2 Identification of adult helminths 146 4.5 Techniques for concentrating parasites 152 4.5.1 Flotation technique using sodium chloride solution (Willis) 152 4.5.2 Formaldehyde–ether sedimentation technique (Allen & Ridley) 153 4.5.3 Formaldehyde–detergent sedimentation technique 154 4.5.4 Sedimentation technique for larvae of Strongyloides stercoralis (Harada–Mori) 156 4.6 Chemical test for occult blood in stools 157 4.6.1 Principle 157 4.6.2 Materials and reagents 157 4.6.3 Method 158 4.6.4 Results 159 4.7 Parasites of the blood and skin 159 4.7.1 Filariae 159 4.7.2 Plasmodium spp. 172 4.7.3 Trypanosoma spp. 182 4.7.4 Leishmania spp. 194 5. Bacteriology 197 5.1 Introduction 197 5.2 Preparation and fixation of smears 197 5.2.1 Principle 197 5.2.2 Materials and reagents 197 5.2.3 Preparation of smears 198 5.2.4 Fixation of smears 199 5.3 Staining techniques 199 5.3.1 Gram staining 199 5.3.2 Staining with Albert stain (for the detection of Corynebacterium diphtheriae) 201 5.3.3 Staining with Ziehl–Neelsen stain (for the detection of acid-fast bacilli) 202 5.3.4 Staining with Wayson stain (for the detection of Yersinia pestis) 203 5.3.5 Staining with Loeffler methylene blue (for the detection of Bacillus anthracis) 204 5.4 Examination of sputum specimens and throat swabs 204 5.4.1 Materials and reagents 205 5.4.2 Method 205 5.4.3 Microscopic examination 206 5.4.4 Dispatch of specimens for culture 206 vi Manual of basic techniques for a health laboratory 5.5 Examination of urogenital specimens for gonorrhoea 207 5.5.1 Materials and reagents 207 5.5.2 Method 207 5.5.3 Microscopic examination 208 5.5.4 Dispatch of specimens for culture 209 5.6 Examination of genital specimens for syphilis 209 5.6.1 Materials and reagents 210 5.6.2 Method 210 5.6.3 Microscopic examination 211 5.7 Examination of semen specimens 211 5.7.1 Materials and reagents 211 5.7.2 Method 212 5.7.3 Macroscopic examination 212 5.7.4 Microscopic examination 212 5.8 Examination of vaginal discharge 215 5.8.1 Materials and reagents 215 5.8.2 Method 215 5.8.3 Microscopic examination 215 5.9 Examination of watery stool specimens 216 5.9.1 Materials and reagents 216 5.9.2 Method 216 5.9.3 Microscopic examination 216 5.9.4 Dispatch of specimens for culture 216 5.10 Examination of aspirates, exudates and effusions 218 5.10.1 Materials and reagents 218 5.10.2 Method 218 5.10.3 Microscopic examination 219 5.11 Examination of pus for Bacillus anthracis 219 5.11.1 Materials and reagents 219 5.11.2 Method 220 5.11.3 Microscopic examination 220 5.12 Examination of skin smears and nasal scrapings for Mycobacterium leprae 220 5.12.1 Materials and reagents 220 5.12.2 Method 221 5.12.3 Microscopic examination 223 6. Mycology 225 6.1 Examination of skin and hair for fungi 225 6.1.1 Materials and reagents 225 6.1.2 Method 225 6.2 Examination of pus for mycetoma 226 6.2.1 Materials and reagents 227 6.2.2 Method 227 6.3 Examination of skin for pityriasis versicolor 227 6.3.1 Materials and reagents 227 6.3.2 Method 228 Contents vii PART III 231 7. Examination of urine 233 7.1 Collection of urine specimens 233 7.1.1 Types of urine specimen 233 7.1.2 Preservation of urine specimens 234 7.2 Examination of urine specimens 234 7.2.1 Appearance 234 7.2.2 Testing for the presence of blood 234 7.2.3 Measuring the pH 235 7.2.4 Detection of glucose 236 7.2.5 Detection and estimation of protein 236 7.2.6 Detection of ketone bodies 239 7.2.7 Detection of abnormal elements 240 7.2.8 Detection of Schistosoma haematobium infection 249 7.2.9 Detection of bacteria 251 8. Examination of cerebrospinal fluid (CSF) 255 8.1 Common reasons for investigation of CSF 255 8.2 Collection of CSF specimens 255 8.3 Examination of CSF specimens 255 8.3.1 Precautions 255 8.3.2 Direct examination 256 8.3.3 Microscopic examination 257 8.3.4 Determination of glucose concentration 261 8.3.5 Determination of protein concentration 262 8.3.6 Summary 263 8.4 Dispatch of CSF specimens for culture 263 8.4.1 Materials and reagents 263 8.4.2 Method using Stuart transport medium (for the isolation of Neisseria meningitidis) 264 9. Haematology 265 9.1 Types of blood cell 265 9.1.1 Erythrocytes 265 9.1.2 Leukocytes 265 9.1.3 Thrombocytes 266 9.2 Collection of blood specimens 267 9.2.1 Principle 267 9.2.2 Materials and reagents 267 9.2.3 Method 267 9.3 Estimation of the haemoglobin concentration 271 9.3.1 Haemiglobincyanide photometric method 271 9.3.2 Alkaline haematin D method 276 9.4 Estimation of the erythrocyte volume fraction 279 9.4.1 Micro-scale method 280 9.4.2 Macro-scale method 286 9.5 Estimation of the erythrocyte number concentration 287 viii Manual of basic techniques for a health laboratory 9.6 Estimation of the leukocyte number concentration 288 9.6.1 Principle 288 9.6.2 Materials and reagents 288 9.6.3 Method 289 9.6.4 Results 291 9.7 Measurement of the erythrocyte sedimentation rate 292 9.7.1 Principle 292 9.7.2 Materials and reagents 292 9.7.3 Method 292 9.7.4 Results 293 9.8 Measurement of the bleeding time: Duke method 295 9.8.1 Principle 295 9.8.2 Materials and reagents 295 9.8.3 Method 295 9.8.4 Results 296 9.9 Observation of clot retraction and measurement of lysis time 297 9.9.1 Principle 297 9.9.2 Materials 297 9.9.3 Method 297 9.9.4 Results 298 9.10 Preparation and staining of thin blood films 299 9.10.1 Principle 299 9.10.2 Materials and reagents 299 9.10.3 Method 300 9.10.4 Microscopic examination 305 9.11 Test for sickle-cell anaemia 314 9.11.1 Principle 314 9.11.2 Materials and reagents 314 9.11.3 Method 315 9.11.4 Microscopic examination 315 9.12 Determination of the reticulocyte number concentration/fraction 316 9.12.1 Principle 316 9.12.2 Materials and reagents 316 9.12.3 Method 317 9.12.4 Microscopic examination 318 9.13 Determination of the leukocyte type number fraction 319 9.13.1 Principle 319 9.13.2 Materials 319 9.13.3 Microscopic examination 320 9.14 Determination of the thrombocyte number concentration 321 9.14.1 Materials 321 9.14.2 Microscopic examination 321 10. Blood chemistry 322 10.1 Estimation of glucose concentration in blood: o-toluidine method 322 10.1.1 Principle 322 10.1.2 Materials and reagents 322 Contents ix 10.1.3 Method 322 10.1.4 Results 324 10.2 Estimation of urea concentration in blood: diacetyl monoxime/ thiosemicarbazide method 325 10.2.1 Principle 325 10.2.2 Materials and reagents 325 10.2.3 Method 326 10.2.4 Results 327 11. Immunological and serological techniques 328 11.1 Introduction to immunology 328 11.1.1 Antibodies 328 11.1.2 Antigens 329 11.1.3 Antigen–antibody interactions 330 11.2 Principle of immunochemical techniques 330 11.2.1 Primary binding tests 330 11.2.2 Secondary binding tests 332 11.3 Determination of rheumatoid factors by the latex-agglutination technique 336 11.3.1 Materials and reagents 336 11.3.2 Method 336 11.4 Tests for the determination of anti-streptolysin O antibodies 336 11.4.1 Anti-streptolysin O test (ASOT) 336 11.4.2 Latex agglutination 338 11.5 Determination of b-human chorionic gonadotropin (b-hCG) in urine by the agglutination inhibition technique 339 11.5.1 Materials and reagents 339 11.5.2 Method 339 11.6 Quantitative determination of IgA, IgG and IgM by radial immunodiffusion 339 11.6.1 Materials and reagents 339 11.6.2 Method 340 11.7 Tests for the determination of HIV antibodies 341 11.7.1 ELISA 341 11.7.2 Dipstick test 342 11.8 Tests for hepatitis virus infection 342 11.8.1 ELISA for hepatitis B surface antigen 343 11.8.2 Dipstick test for hepatitis B surface antigen 344 11.9 Dipstick test for falciparum malaria 344 11.9.1 Materials and reagents 344 11.9.2 Method 345 11.10 Tests for syphilis infection 346 11.10.1 RPR test 347 11.10.2 TPHA test 348 Annex: Reagents and their preparation 350