280 Index. Bacteriophage typing, discrimination, 18 reaction difference rule, 19, 23, 24 reproducibility, 18, 19 selection of bacteriophage, 17

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1 Index Antibiotic, 1-4 factors affecting activity, 3 minimum inhibitory concentration (MIC), 1, 3 solubility, 2, 4 Antibiotic resistance profiling, 1-4 antibiogram, 2 breakpoint method, 2 Antibodies, enzyme labels, 123, 127, 135, 138,186 fluorescent conjugated, 107 monoclonal, 108, 124, 125 other labels, 123 polyclonal, 108, 124, 125 secondary, 108 specificity, 108 B Bacterial genomic DNA, , 155, 156, , , additional purification, 146, 196 in agarose plugs, 155, 156 GES method, preparation, , 182, 183, theoretical discussion, 141, 142 total restriction pattern analysis, , 181 Bacteriocin, 5-14 activity spectra, 6 conditions affecting production, 8, 9 indicator strain, 5, 7 mode of action, 6 producer strain, 5, 7 resistance and tolerance, 6 subtypes, 6 Bacteriocin typing, 5-14 active or passive methods, 7 codes, 13 discrimination, 7 lysate method, 7, 10, 11 overlay method, 8-10 production/sensitivity (P/S) typing, 13 reproducibility, 7 scrape and streak method, 7, 11 Bacteriophage, host range, 16, 22 isolation, 17, 19, 20 from environmental sources, 17, 19 from lysogenic cultures, 20 life cycle, 15, 16 lysogeny, 15 lytic, 15 plaque, 16 temperate, 15 virulent, 15 propagation, 22 purification, 21 storage, 18, 24 titer, 20, 21, 24, 25 routine test dilution (RTD), 21, 23,25 279

2 280 Index Bacteriophage typing, discrimination, 18 reaction difference rule, 19, 23, 24 reproducibility, 18, 19 selection of bacteriophage, 17 Carboxylic acids, 80 Cell lysis, acetone, 37 alkaline-sds, , boiling, 241, 244, 261 guanidinium isothiocyanate (GIC), 143, 183, 188 lysostaphin, 37, 146 lysozyme, 30, 37, 146, 182, 188, , 229 mutanolysin, 146, 183, 188 NaOH, 135, 138 in PCR mix, 251,254 sarkosyl, 143, 183, 188 in agarose plugs, 153, 155,156 SDS, 30, 37, 182, 187 sensitization by growth conditions, 195 sonication, 37, 54, 60, 70, 77 Chemiluminescence, see Horseradish peroxidase Chemotype, 43 Computer software, analysis of DNA patterns, 175,176 databases, 83 PYMENU, 98, densitometry, 169 DNA sequence analysis, 249, 252 for primer design, 254, 257, 276 statistical analysis, 83, 169,174, 175 Curie point, D DEFT, see Direct epifluorescent filter technique Densitometry, 34, 169, 174 protein gels, 34 DIPT, see Direct immunofluorescent filter technique Direct epifluorescent filter technique (DEFT), automation, 116 correlation with total viable counts, 114, 115, 119 fluorescent stain, 114, 115 microcolony method, 115 pretreatment with enzyme/ detergent, 114 as a rapid method, 113 sensitivity, 113, 114 use of Nuclepore filters, 114 Direct immunofluorescent technique (DIPT), as a rapid method, 108 sensitivity, 109 DNA, estimation by absorbance at 260 nm, 145, 189, 210, 261 estimation of concentration, estimation by ethidium bromide staining and visual comparison, 220 estimation by fluorometry, 205, 206, 210 fragment purification with GENECLEAN, 203, 205, 209, 210 E ELISA, 123, 134 commercial kits, 127 competitive, 126 noncompetitive, 126, 127 double antibody sandwich, 127 sensitivity, 126, 127 Enzyme-linked immunosorbent assay, see ELISA Epifluorescent microscopy, ,114

3 Index 281 FAB-MS, see Mass spectrometry H Horseradish peroxidase, chemiluminescent detection, 186, 187, 193, 194, 208, 209 colorimetric assay, 130, 138, 139 labeling, of DNA, 203, of rrna, 182, 186 I Immunoassay, 123 Immunoenzymatic detection, see Horseradish peroxidase Irradiated foods, detection of, 115 LCR, see Ligase chain reaction Ligase chain reaction, analysis of products, 273, 274 buffers for, 271,272,276 limits of detection, 271 oligonucleotide, design, 269, 270 labeling using TdT, 272 precautions against contamination, 275 quantitation of results by liquid scintillation counting, 273, 274 use of master mix, 272 Ligase, thermostable, ,273,276 template independent ligation, 271 unit definition, 276 Lipid extraction, from bacteria, 84 Lipopolysaccharide, constituents of, 41 Lipopolysaccharide chemotyping, LPS, see Lipopolysaccharide Luminol, see Horseradish peroxidase, chemiluminescent detection Lysostaphin, see Cell lysis Lysozyme, see Cell lysis M Mass spectrometer, 83, 98, 99 Mass spectrometry (MS), 79-95, chemical ionization, 79 electron impact, 79 fast atom bombardment (FAB- MS), data analysis, identification of compounds, 87 parameters, 93 of polar lipids, 80 pyrolysis (PyMS), data analysis, in outbreak epidemiology, 102 in taxonomic studies, 103 MEE, see Multilocus enzyme electrophoresis Mitomycin C, induction of bacteriocins, 9, 11 induction of bacteriophage, 20 Molecular weight markers, DNA, 184, 203 circular, 216 internal, 169, 170, 174 for PFGE, 154, 155 protein, 32, 57 Multilocus enzyme electrophoresis (MEE), electromorph, 64 electrophoretic type (ET), 64 Mutanolysin, see Cell lysis O 0-antigen, see Lipopolysaccharide

4 282 Index Outer membrane proteins (OMPs), constitutive, 49 heat modifiable, 56, 58 inducible, 49, 58 major (MOMPs), 58 membrane-associated protein structures, 49, 55 peptidoglycan associated, 56, 58 pore forming proteins (porins), 49, 56 Outer membranes, preparation of, 53 PCR, , , 269 for bacterial identification, for development of species specific probes, primer design strategy, 247, 249 multiplex, 239, 241 precautions against contamination, 241, 244, 245, 250, 254, 265 oligonucleotide design, calculation of Tm, 253, 254, 257 species-specific, , 241, 245, 253 sequencing of products, 251, 252 use of pre-mix, 244, 251, PFGE, see Pulsed field gel electrophoresis Phenylmethylsulfonyl fluoride, see PMSF Phospholipids, 81 Photography, alternatives to, 184, 194, 262 DNA, 184,220,262 silver stains, 45 Plasmid DNA preparation, , cesium chloride density gradient centrifugation, modified Birnboim and Doly method, 226, modified Kado and Liu method, 217,218,227,231,232 modified Portnoy method, 214, native plasmids, 213, 214 Plasmid fingerprinting, Plasmid profiling, PMSF, 61, 154 Polymerase chain reaction, see PCR Probes, nonradioactive, , 181, acetylaminofluorine (AAF) labeled RNA, GeneTrak kit, capture probe, 135, 138 detector probe, 135, 138 dipstick, sensitivity, 133 specificity, 134 horseradish peroxidase labeled, see also Horseradish peroxidase DNA, 203, rrna, 182, 186, 193, 194 oligonucleotide, species-specific, 108, 134, 135, 253 Protein quantitation by Lowry method, 53, 56, 57 Pulsed field gel electrophoresis (PFGE), applications, 152 electrophoresis conditions, 160 factors affecting DNA mobility, 151 size limit, 149 variations, 149, 150 PyMS, see Mass spectrometry R Random Applied Polymorphic DNA, see RAPD

5 Index 283 RAPD, factors affecting, PCR parameters, 262 primer design, 259, 260 related methods, 264 storage of reagents, 264, 265 theoretical discussion, Restriction endonuclease digestion, , 168, 170, 183, 189, 229, in agarose, digestion with a single enzyme, 156 partial digestion, 156, 161 for two dimensional PFGE, 157 Ribosomal RNA (rrna) genes, , 247 restriction patterns of, Ribotyping, see Ribosomal RNA (rrna) genes rrna genes, see Ribosomal RNA (rrna) genes SDS-PAGE, bacterial protein patterns, background pattern, 34 major band region, 34 of digested DNA, 165, 168, electrophoresis parameters, 32 of lipopolysaccharide, 43, 44 of outer membrane proteins, 49, 57 reproducibility, 28, 32, 35 smiling, 32 Serotype, 43 Somatic antigen, see Lipopolysaccharide Southern blotting, carrier DNA, 185 hybridization, 181, 185 of horseradish peroxidaselabeledprobes, 208, 211 probes for, 181 vacuum transfer, 184, 190, 191 Staining, Coomassie blue, 30, 32, 33 enzyme activity, 63, 66-70, agarose overlay, 72, 78 silver, DNA, 165, 167, 169, 172 lipopolysaccharide, 43, 45 Starch gels, 64 interpretation of activity stains, 75 preparation, 70, 77 Total viable counts (TVCs), 113