2016 UNIVERZITA KOMENSKÉHO V BRATISLAVE JESSENIOVA LEKÁRSKA FAKULTA V MARTINE Jana KOMPANÍKOVÁ Martina NEUSCHLOVÁ Vladimíra SADLOŇOVÁ SPECIAL BACTERIOLOGY BASIC LABORATORY TESTS Preface Special Bacteriology – Basic Laboratory Tests is intended above all for medical students. The book includes standard procedures commonly used in microbiological laboratory. We have tried to present principles of laboratory tests to make them easier to understand. Authors Contents 1 STAPHYLOCOCCI ........................................................................................................ 5 1.1 GRAM STAIN ................................................................................................................... 7 1.2 STAPHYLOCOCCI - BLOOD AGAR CULTURE .................................................................... 7 1.3 CATALASE TEST ............................................................................................................... 8 1.4 MANNITOL SALT AGAR CULTURE ................................................................................... 9 1.5 COAGULASE TEST ......................................................................................................... 11 2 STREPTOCOCCI ........................................................................................................ 14 2.1 STREPTOCOCCI - GRAM STAIN ..................................................................................... 15 2.2 STREPTOCOCCI - BLOOD AGAR CULTURE ..................................................................... 16 2.3 STREPTOCOCCI - SEROLOGIC PROPERTIES TESTING ..................................................... 18 2.4 STREPTOCOCCI - PHYSIOLOGIC (BIOCHEMICAL) PROPERTIES TESTING ...................... 20 2.5 ANTISTREPTOLYSIN O (ASO) ......................................................................................... 21 2.6 C-REACTIVE PROTEIN (CRP) .......................................................................................... 22 3 NEISSERIA ............................................................................................................... 23 3.1 NEISSERIA - GRAM STAIN ............................................................................................. 23 3.2 NEISSERIA - BLOOD AGAR CULTURE ............................................................................. 24 3.3 NEISSERIA - CHOCOLATE AGAR CULTURE .................................................................... 25 3.4 NEISSERIA - BIOCHEMICAL PROPERTIES TESTING ........................................................ 26 4 HAEMOPHILUS ........................................................................................................ 30 4.1 HAEMOPHILUS - GRAM STAIN ..................................................................................... 31 4.2 HAEMOPHILUS – CULTIVATION ON BLOOD AGAR ....................................................... 32 4.3 HAEMOPHILUS - CULTIVATION ON CHOCOLATE AGAR .............................................. 32 4.4 X AND V FACTOR DISKS FOR DIFFERENTIATION OF HAEMOPHILUS SPECIES............... 33 4.5 QUELLUNG TEST ........................................................................................................... 35 5 ENTEROBACTERIACEAE ............................................................................................ 36 5.1 ENTEROBACTERIACEAE - GRAM STAIN ........................................................................ 37 5.2 BURRI´S INDIA INK METHOD (CAPSULE VISUALISATION) ............................................ 39 5.3 ENTEROBACTERIACEAE - CULTIVATION ON BLOOD AGAR........................................... 40 5.4 ENTEROBACTERIACEAE – CULTIVATION ON ENDO AGAR ............................................ 42 5.5 ENTEROBACTERIACEAE – CULTIVATION ON DEOXYCHOLATE CITRATE AGAR ........... 43 5.6 ENTEROBACTERIACEAE- BIOCHEMICAL PROPERTIES - TRIPLE SUGAR IRON AGAR (TSI, HAJN) .................................................................................................................... 44 5.7 UREASE TEST ................................................................................................................. 46 5.8 SIMMON´S CITRATE AGAR............................................................................................ 46 5.9 WIDAL REACTION ......................................................................................................... 47 5.10 BACTERIAL SEROTYPING ............................................................................................. 48 5.11 ENTEROTEST - PRINCIPLE ............................................................................................ 49 6 CAMPYLOBACTER, HELICOBACTER ........................................................................... 51 6.1 CAMPYLOBACTER JEJUNI .............................................................................................. 51 6.2 CAMPYLOBACTER - GRAM STAIN ................................................................................. 51 6.3 CAMPYLOBACTER - CULTIVATION ................................................................................ 51 6.4 HELICOBACTER PYLORI ................................................................................................. 52 6.5 HELICOBACTER - GRAM STAIN ..................................................................................... 52 6.6 HELICOBACTER - CULTIVATION .................................................................................... 53 6.7 UREASE TEST ................................................................................................................. 53 6.8 UREA BREATH TEST ...................................................................................................... 54 6.9 HELICOBACTER HEILMANNII ........................................................................................ 54 7 CORYNEBACTERIA ................................................................................................... 56 7.1 CORYNEBACTERIUM DIPHTERIAE ................................................................................. 56 7.2 CORYNEBACTERIA - GRAM STAIN................................................................................. 57 7.3 CORYNEBACTERIA – ALBERT STAIN (METACHROMATIC GRANULES) ......................... 58 7.4 CORYNEBACTERIA – CULTIVATION ON BLOOD AGAR ................................................. 59 7.5 CORYNEBACTERIA – CULTIVATION ON TELLURITE AGAR AND TINSDALE AGAR ......... 59 7.6 ELEK´S TEST ................................................................................................................... 60 8 BORDETELLA ........................................................................................................... 64 8.1 BORDETELLA PERTUSSIS ............................................................................................... 64 8.2 PERTUSSIS - SAMPLING ................................................................................................ 65 8.3 BORDETELLA PERTUSSIS – GRAM STAIN ...................................................................... 66 8.4 BORDETELLA PERTUSSIS – CULTIVATION(BORDET GENGOU BLOOD AGAR, CHARCOAL HORSE BLOOD AGAR) .................................................................................................. 66 9 ANAEROBIC BACTERIA .......................................................................................... 9-69 9.1 CLOSTRIDIA ................................................................................................................ 9-71 9.2 LABORATORY DIAGNOSIS OF ANAEROBIC BACTERIA ................................................ 9-73 9.3 ANAEROBIC INFECTIONS – SPECIMEN COLLECTION.................................................. 9-74 9.4 ANAEROBIC BACTERIA - GRAM STAIN ....................................................................... 9-74 9.5 ANAEROBIC BACTERIA – WIRTZ - CONKLIN STAIN .................................................... 9-77 9.6 ANAEROBIC BACTERIA - CULTIVATION ...................................................................... 9-78 10 SPIROCHETES .......................................................................................................... 83 10.1 TREPONEMA PALLIDUM ........................................................................................... 84 10.2 SYPHILIS .................................................................................................................... 85 10.3 SYPHILIS – LABORATORY TESTING ............................................................................ 86 10.4 TREPONEMA PALLIDUM - CULTIVATION .................................................................. 86 10.5 TREPONEMA PALLIDUM - MICROSCOPY .................................................................. 86 10.6 TREPONEMA PALLIDUM - SEROLOGICAL TESTS (NONTREPONEMAL AND TREPONEMAL TESTS) ............................................................................................................ 87 11 MYCOBACTERIA ...................................................................................................... 93 11.1 NONTUBERCULOUS MYCOBACTERIA – RUNYON CLASSIFICATION ......................... 93 11.2 MYCOBACTERIUM TUBERCULOSIS (TUBERCULOSIS) ............................................... 94 11.3 MYCOBACTERIUM TUBERCULOSIS – SAMPLE COLLECTION ................................... 96 11.4 MYCOBACTERIUM TUBERCULOSIS – ZIEHL-NEELSEN STAIN .................................... 97 11.5 MYCOBACTERIUM TUBERCULOSIS - CULTIVATION .................................................. 99