APPENDIX-I The compositions of media used for the growth and differentiation of Pseudomonas aeruginosa are as follows: COMPOSITION OF DIFFERENT MEDIA STOCK CULTURE AGAR (AYERS AND JOHNSON AGAR) Gms/Litre Beef Heart infusion from 500 Proteose peptone 10 Gelatin 10 Dextrose 0.50 Caesin purified 5 Disodium phosphate 4 Sodium citrate 3 Agar 7.50 Stock culture agar media was mixed in 1000 ml of distilled water and sterilized by autoclaving at NUTRIENT AGAR Peptic digest of animal tissue 5.0 Sodium chloride 5.0 Beef extract 1.50 Yeast extract 1.50 Agar 15.0 NUTRIENT BROTH Peptic digest of animal tissue 5.0 Sodium chloride 5.0 Beef extract 1.50 Yeast extract 1.50 124
The constituents were suspended in 1L of distilled water and boiled to dissolve and sterilized by autoclaving at PSEUDOMONAS ISOLATION AGAR Peptic digest of animal tissue 20.0 Magnesium chloride 1.4 Potassium sulphate 10.0 Troclosan (Irgasan) 0.025 Agar 13.6 MUELLER HINTON AGAR Beef infusion 300 Caesin acid hydrolysate 17.50 Starch 1.50 Agar 17.00 MOTILITY TEST MEDIUM Tryptose 10 Sodium chloride 5 Agar 5 The specified amounts of tryptose and sodium chloride were added in 1000ml of distilled water and sterilized by autoclaving 125
SIMMONS CITRATE AGAR Gms/Litre Magnesium sulphate 0.20 Ammonium dihydrogen phosphate 1.0 Dipotassium phosphate 1.0 Sodium citrate 2.0 Sodium chloride 5.0 Bromo thymol blue 0.08 Agar 15.0 The constituents were suspended in 1L of distilled water and boiled to dissolve and sterilized by autoclaving at. KING S MEDIUM A BASE Proteose peptone 20.0 Potassium sulphate 10.0 Magnesium chloride, anhydrous 1.64 Agar 15.0 KING S MEDIUM B BASE Proteose peptone No. 3 20.0 Dipotassium hydrogen phosphate 1.50 Magnesium sulphate, 7H 2 O 1.50 Agar 20.0 126
The constituents were suspended in 1L of distilled water and boiled to dissolve and sterilized by autoclaving at. NITRATE BROTH Peptic digest of animal tissue 5.0 Meat extract 3.0 Potassium nitrate 1.0 Sodium chloride 30.0 TRYPTONE SOY AGAR Pancreatic digest of casein 15.0 Papaic digest of soyabean meal 5.0 Sodium chloride 5.0 Agar 15.0 NUTRIENT GELATIN Peptic digest of animal tissue 5.0 Beef extract 3.0 Gelatin 120.0 The constituents were suspended in 1L of distilled water and boiled to dissolve and sterilized by autoclaving at 127
APPENDIX-II The details of chemicals and the reagents used in the study are as follows: BUFFERS AND SOLUTIONS 3% H 2 O 2 H 2 O 2 is prepared by adding 30ml of 30% H 2 O 2 in 70 ml distilled water. Sulfanilic acid solution Sulfanilic acid solution is prepared by dissolving 8g of sulfanilic acid in 1 litre of 5N acetic acid. α-naphthylamine solution α-naphthylamine solution is prepared by dissolving 6g of N,N-Dimethyl-1- naphthylamine in 1 litre of 5N acetic acid. Kovac s oxidase reagent Kovac s oxidase reagent was prepared by dissolving 1g of tetra-methyl-pphenylenediamine dihydrochloride in 100ml of distilled water. 0.5 MacFarland standard BaCl 2 Barium chloride in a vol. of 0.5ml 0.048mol/l (175g of BaCl 2 in 100ml of distilled water) is added to 99.5ml of 0.18mol/l H 2 SO 4 (1ml of H 2 SO 4 in 99ml of distilled water). 1Mol NaOH 40g of NaOH pellets are dissolved in 1000ml of distilled water. Normal saline Normal saline is prepared by adding 0.85 gm of NaCl in 100ml of distilled water. 50% Glycerol Glycerol was prepared by adding 50ml of glycerol in 50ml of distilled water. 70% Ethanol 70% ethanol is prepared by adding 70.0ml absolute ethanol in 30ml of distilled water. 1% Agarose Agarose was prepared by adding 1.0 gm of agarose in 100ml of distilled water. 128
PCR Reagents Lysis Buffer Component 1mM Tris-HCl 10ml 0.5M EDTA 5ml 5% SDS 1ml dh 2 O 100ml TE Buffer Component 1M Tris ph 8 5ml 0.5M EDTA ph 8 1ml dh 2 O 494ml Phenol chloroform isoamyl alcohol mix A mixture of phenol chloroform isoamyl alcohol is prepared in the ratio of 25:24:1. TAE Buffer Component 2M Tris Base 600ml 0.5M EDTA 100ml dh 2 O 300ml Ethidium bromide solution Ethidium bromide solution is prepared by adding10mg ethidium bromide to 1ml distilled water and stored in dark and cool place. Molecular grade water (Gbiosciences, India) 2mM each dntp (Gbiosciences, India) Taq DNA polymerase (5U/µl) (Invitrogen, India) 10X PCR buffer (Invitrogen, India) 1kb marker (Gbiosciences, India) Bromophenol blue 0.25% (w/v)-25mg Primers for various genes (Eurofins Genomics India Pvt Ltd) 129
APPENDIX-III The details of equipment and glassware used in the study are as follows: EQUIPMENT Incubator (Instruments and chemicals pvt.ltd (INCO), India) Laminar Air Flow (Instruments and chemicals Pvt. Ltd (INCO), India) Autoclave (Instruments and chemicals Pvt. Ltd (INCO), India) Weighing balance (Kerro), Thermocycler (AB Biosystems), Gel documentation system (Alpha innotech), U.V. Transilluminator (Cleaver Scientific), Refrigerator (Samsung, India), Deep freezer (New Brunswick Scientific), Centrifuge (Eppendorf), Electrophoresis unit (BenchTop Lab systems). GLASSWARE Petri plates (Genaxy India), Test tubes (Borosil), Conical flasks (100ml, 250ml, 500ml and 1000ml, Borosil), Measuring cylinders (Borosil), Beakers (100ml, 250ml and 500ml, Borosil), Glass slides (Abron instruments) MICELLANEOUS Micropipette (Adarsh, India), Inoculation loops (Himedia), Non-absorbent cotton, Filter paper, Aluminium foil, Microtips (Genaxy), Paper discs (Himedia). 130
APPENDIX-IV Zone Size Interpretative Chart Sr Antibacterial Agent Sensitive Intermediate Resistant No 1 Amikacin 17 15-16 14 2 Gentamicin 10 7-9 6 3 Cefepime 18 15-17 14 4 Ceftazidime 18 15-17 14 5 Ciprofloxacin 21 16-20 15 6 Piperacillin/Tazobactum 21 15-20 14 7 Imipenem 19 16-18 15 8 Meropenem 19 16-18 15 9 Levofloxacin 17 14-16 13 10 Aztreonam 22 16-21 15 11 Piperacillin 21 15-20 14 12 Cefoperazone 21 16-20 15 13 Colistin 11-10 14 Tobramycin 15 13-14 12 15 Carbenicillin 17 14-16 13 16 Polymixin B 12-11 17 Gatifloxacin 18 15-17 14 18 Cefazolin 23 20-22 19 19 Azthithromycin 18 14-17 13 20 Tigecycline 9-13 21 Ticarcillin/Clavulanic 24 16-23 15 acid 22 Ofloxacin 16 13-15 12 23 Netillin 15 13-14 12 24 Cefotaxime 23 15-22 14 25 Doripenem 19 16-18 15 26 Etrapenem 13-21 131