AN NEXURE. B log Sodium chloride 5g Distilled water (DW) 1 Litre ph: ] g 100 ml pl-l: g Glucose

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1 AN NEXURE A. Composition of bacteriological media l. Alkaline Water (APW) B log Sodium chloride 5g Distilled water () 1 Litre ph: ] 2. Brilliant Green Bile Broth (BGLB ) Bile salt Brilliant green g pl-l: Decarboxylase Broth Basal medium Yeast extract Glucose Bromocresol purple (BCP) 0.00l6g 100ml ph: 6.5 1; 0.1 One amino acid to 100ml of the basal medium. l.l-lysine Hydrochloride 2. L-Arginine Hydrochloride 3. L-Ornithine I-lydrochloride 4. DNAse with Toluidine blue Tiyptose DNA Toluidine blue Distilled water 20g 5%; 22 llitre ph: 7.3 i 0.2

2 5. EC broth 6. Egg Yolk Tryptone Bile salt (No.3) KHZPO4 Nutrient '7. Eosin Methylene Blue ) Zr; 0.5 g 0.l5 g 0.4 g 0.l5g 0.5 g ph: 6.9 i0.l l00ml 50% Egg Yolk solution Sml ph 7i 0.2 Eosin Y Methylene blue l0g 22; 0.4g 0.065g ph: 7.1 i Fish powder agar Beef extract Fish powder l0g 3g 30g 15 g 1 litre ph: 7.0 i 0.1 Fish powder agar with different salt concentrations was prepared by adding the required quantity of to the media along with all other ingredients before autoclaving. 9. Gelatin KHZPO4 Nutrient l000ml Gelatin 4 l.5g Glucose 0.05 gg ph

3 10. Gelatin Medium l Og Beef extract 3 g Gelatin 5 s I20 g i j 7 p_h:7.0#0.l _ fi ll. Hugh & Leifson Glucose O/F Medium (H&L) N acl KQHPOJ Dextrose 0.4g is l0oml ph: ,1 Add ll ml of0. 1% solution of phenol red indicator I2. Kligler Iron (KIA) Yeast extract Beef extract Glucose Ferric citrate Sodium thiosulphate Phenol red 20g 32 3g g 1 litre ph 7.4 io Methyl Red Vogues Prausker Medium (MRVP) 14. Nitrate Broth D-glucose Potassium nitrate 100ml pl-l: 6.9 i 0.1 K o l0oml ph: 7.1 i 0.1 _

4 IS. Mueller Hinton Casein acid hydolysate 17.5g Beef infusion 300g Starch 17g 1.5g 1000ml _ ph Normal Saline (NS) NaC1 (Physiological 85g Saline) N I7. Nutrient I8. Nutrient Broth _ 1 litre Beef extract 3g 5 g l l5 litre g h pl-l: 7.Qi 0.1 Beef extract 3g 5 g 1 litre % ph: 7.0 i ONPG (O-nitrophenyl galactopyranoside) ONPG solution : onvo i 6g Na2HPO4 buffer l000ml Dissolve and filter N it water: A 1\IaCl 5 g 1000ml Test: Take 1 part ofon PG solution and 3 parts of peptonie water in small tubes inoculate heavily with the test culture. lncubate at 37 C. Read after 20 min to 24h. Yellow colour indicates positive result P1 N1) Medium N Distilled water 100ml * _ ph: 7.0 i Q.l 291

5 21. Phosphate Buffer Diluent KHZPO4 34g Distilled water 500ml Adjust ph to with 1 N l\laoh and bring volume to 1 litre with distilled water. Take 1.25 ml of the above stock solution and bring volume to 1 litre with distilled water. Dispense in flasks or tubes and sterilize at 121 C for 15min. 22. Purple Broth Base (For Sugar Fermentation) NaC1 5 g Bromocresol purple 0.02 g M ph to 7.0 i 0.2 Add the required. sugar to 1% level. 23. Shrimp powder agar Beefextract 3g Shrimp powder 1530g 1 litre g A ph: 7.0i0.l g g Shrimp powder agar with different salt concentrations was prepared by adding the required quantity of to the media along with all other ingredients before autoclaving. 24. Simmon s Citrate Sodium citrate 0.2 g N ac g l\lh4h2po4 MQSO4 Bromothymol blue 25. Skim milk agar g g Casein enzymatic hydrolysate Yeast Extract Dextrose Skim milk powder 0.02g 0.008g 1.5g _g ph: 7.0 i 0.2 5g 2.5g ls 28g g_ i pkl g 92

6 26. Starch Beef extract NaCJl Soluble Starch l0g -is 5 g ph: 7.0i().l (_ 27. Sugar Fermentation Media 28. Tergitol-7 (T-7) Sodium chloride 10 g 5 g ph: 7.2 i 0.1 Add 10ml of phenol red indicator, adjust ph. Add the required sugar to 1% level. Yeast extract Beef extract Tergitol-7 Bromothymol blue g 0.05g ph: 7.2 ; : 0;; H Before pouring the plates, after melting and cooling, add 0 25ml of 1% solution of sterile Triphenyl Tetrazolium C hloiide (TTC ) per media. 29. a) Thiosulfate Citrate Bile Salt Sucrose (T CBS) Yeast extract Sucrose Sodium thiosulphate Sodium Citrate Dihydrate Sodium cholate Ox-gall Sodium chloride Ferric citrate 5g C 20g 3s 52 l0g Bromothymol blue (BTB) 0.04g Thymol blue (TB) 0.04g 976ml ph: 8.6 :t 0.1

7 I1) TCBS (Difco) Formula Per Liter Bacto Yeast Extract 5 g Bacto Proteose No. 3 I0 g Sodium Citrate I0 g Sodium Thiosulfate l0 Bacto Oxgall 8 Bacto Sac-charose 20 g Sodium Chloride l0 g Ferric Citrate 1 g Bac-to Bromo Thymol Blue 0.04 g Thymol Blue 0.04 g Bacto 15 g W g Final ph 8.6 i 0.2 at 25 C, 30. T1N0 and T1 N 3 Medium Tiypticase ml g ph: i 0.2 Dissolve; adjust the ph; divide into two lots. To one lot add 3 g, dissolve and dispense in 5 ml quantities. This is TIN; medium. The second lot is dispensed in 5 ml quantities in test tubes. This is TING medium. 31. Tributyrin agar g M Peptic digest of animal tissue 5 g 32. Triple Sugar Iron (TSI) Yeast Extract 3 g 1000ml _n_ ph 7.5 i0.g2 Add 10ml of Trilautyrin (glycerol tributyrate) to of the medium. 20g Yeast extract 3g Beef extract 3 g Sucrose 5g Glucose Ferric citrate Sodium thiosulfate Phenol red (0.2% soln:) I2 ml 12g 988 ml ph: 7.4 i

8 H. Tryplicase Salt Broth l L. Tiyipticase M ph: 7.2 i Add IO, , 80 and 100g, as the case may be to make media with salt concentration of 3, 6, 8 and 10% Distnbute 5 ml quantities In test tubes. 34. Tryptone Broth (lntlole Medium) Tiyptonc N acl 1 g 0.5 g ph 7.l_:0,l 35. Tryptone Glucose (TGA) Tiyptone Beef Extract (Lab lemco) D-Glucose agar Distilled wate1'(_) 36. Tryptone Soy (Difco M) %_ T Pancreatic digest of casein Soybean peptone PW l.5g ph: ] 52 5s 1000ml 37. Urea Dextrose Sodium Chloride Potassium di hydroge phosphate Phenol red Distilled water 52 n 2g 0.0l2g 1000ml ph: 6.8 {(11 /itter sterilizing:,cool to 50 C and aseptically add 0 5 ml of a 40 % solution ofurea per tube, mix well and allowed to cool and fonn slants.

9 B. Test Reagents 1. Kovac s cytochrome oxidase reagent N:N:N:N.Tetramethyl-p-phenylene 100mg diamine -hydrochloride 10ml 2. Kovac s lndole reagent p-dimethyl amino benzaldehyde N-butyl alcohol (or amyl alcohol) 52;. 75nfl 3. VP test reagents and test Solution A 01-naphthol Alcohol g W Solution B 7 KOH 0.25g Sml _ ow 22; 5 ml 4. Reagents for nitrate reduction test Solution A 0t- Naphthylamine 0.5 g Dilute Sulphuric Acid (f "I :20 ) g mm Dissolve, filter and store in a brown bottle..q l~1-till-3) S 77 Sulphanilie Acid 0.8 g Dilute Sulphuric Acid (l:l25 i) loq ml 5. Mercuric chloride solution _ HCI 20ml Dw l00ml