Tryptone Water                       Tryptone                                     10 gm         Sodium chloride                           5 gm         Distilled water                            1000 ml         Dissolve the solids by stirring in thewater bath. Leave the reaction unadjusted          and sterilize at 15 psi pressure 121°C temperature for 20 minutes.         GlucosePhosphate Peptone Water (GPPW)       Glucose                                                         5.00 gm         Peptone                                                         5.

00 gm         Dipottassiumhydrogen Phosphate               5.00 gm         Distilled Water                                             1000.00 ml            Sterilize by autoclaving at 15 psipressure, 121° C temperatures for 30 minutes.         LuriaAgar         Compositionper liter:         Agar                                                      15.

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0 gm         Pancreatic digest of casein                    10.0 gm         Yeast extract                                           5.0gm         Nacl                                                        0.5 gm         Glucose solution                                      20.0ml           pH7.0 ± 0.2 at 25° C         Simmons’Citrate Agar                                                  (Dehydrated, Hi Media)          Ingredients                                                                                        Grams/liter         Magnesium sulphate                                                                             0.20              Ammonium dihydrogen  phosphate                                                       1.

00             Dipotassium phosphate                                                                         1.00             Sodium citrate                                                                                       2.00             Sodium chloride                                                                                    5.

00             Bromo thymol blue                                                                               0.08             Final Ph (at 25° C) 6.8 ± 0.2                                                     Preparation of Medium Suspended24.28 gm in 1000 ml distilled water, distributed in test tube and sterilized by autoclaving at 15 psi pressure, 121° Cfor 20 minutes.            Luria Broth          Composition perliter:            Pancreatic digest of casein                                                                        10.0 gm            Nacl                                                                                                            5.0 gm            Yeast extract                                                                                               5.

0 gm            Glucose                                                                                                       1.0 gm          Preparation of Medium          Add components todistilled/ deionized water and bring volume to 1 liter. Mix thoroughly.           Distribute into tubes or flasks.Autoclave for 15 minute at 15 psi pressure 121° C.            Use:for the cultivation of Escherichia coli& Klebsiella pneumonia.             Mueller Hinton (MH) Agar                                                 (Dehydrated, Hi Media)             Ingredients                                                                              Grams/liter            Casein acid hydrolysate                                                               17.50            Beef heart infusion                                                                       2.

00            Starch, soluble                                                                               1.5           Agar                                                                                            17.00 gm            FinalpH(at 25° C) 7.3 ± 0.2           Preparationof Medium           Suspended 38 gm in 1000 ml distilledwater.

Sterilized by autoclaving at 15 psi pressure,            121° C for 20 minutes. The molten medium wascooled to about 50° C temperature and            poured into sterile petriplates.          GlucoseSolution         Compositionper 100 mL         Glucose……………………………………………………………….

.10.0g           Preparation of Glucose Solution              AddGlucose to distilled/deionized water and bring volume to 100 mL. Mixthoroughly.          Filtersterilize.          Preparationof Medium         Add components,except glucose solution, to distilled water and bring Volume to 980 mL.

               Mixthoroughly. Bring pH to 7.0. Gently heat and bring to boiling. Autoclave for 15min at           15 psi pressure 121° C.

Aseptically add 20 mLof sterile glucose solution. Mix thoroughly.         Pour into sterile Petridishes or leave intubes.          Use:For the cultivation of Escherichia coli & Klebsiella pneumonia.

         TryptoneBroth        Compositionper liter         Pancreatic digest of casein                                          10.og         Nacl                                                                                5.0g         pH 7.5 ± 0.2 at 25° C.

         Preparationof Medium:        Addcomponents to distilled/deionized water and bring volume to 1 L. Mixthoroughly.          Distributeinto tubes or flasks. Autoclave for 15 min at 15 psi pressure 121° C.        Use: For the cultivation of  production of indole by microorganisms.        Citrate Agar        Composition perliter:                                         Agar                                                                            15.0g        Nacl                                                                             5.

0g        Sodium citrate                                                             2.0g        K2HPO                                                                       41.0g        (NH4)H2PO                                                              41.

0g               MgSO4.7H20                                                             0.2g        BromthymolBlue                                                      0.08g        pH 6.

9 ± 0.2 at 25° C        Preparation of Medium:       Add components todistilled/deionized water and bring volume to 1 L. Mix thoroughly.            Gentlyheat while stirring and bring to boiling.

Distribute into tubes or flasks.Autoclave for         15min at 15 psi pressure 121° C. Pour into sterile Petridishes or leave in tubes.        Use:For the differentiation of Gram-negative bacteria on the basis of citrateutilization.         Bacteriathat can 394 Skim Milk Agar utilize citrate as sole carbon source turn themedium         blue.       MacConkeyAgar (MCA)     Ingredients                                                                              Grams/litre      Peptic digest of animal tissue                                                    20.00 gm      Lactose                                                                                       10.00 gm      Bile salt                                                                                         5.

00gm      Sodium chloride                                                                           5.00 gm      Neutral red                                                                                   0.07 gm      Agar                                                                                            15.00 gm      Distilled water                                                                           1000.

00 ml      Final pH (at 25° C) 7.5 ± 0.2      Preparationof Medium:     Suspended 55.07 gmof dehydrated MCA in 1000 ml distilled water and sterilized by       autoclaving at 15 psi pressure, 121° Cfor 20 minutes.

The molten medium was cooled to       about 50° C temperature and poured intosterile petriplates.      Use:For the detection of members of the Enterobacteriaceae and Enterococci aswell                                                                                                                                                                                                                                                                        assome staphylococci.      EosinMethylene Blue (EMB) Agar      Ingredients                                                                       Grams/liter                                                                             Peptone                                                                                10.00       Lactose                                                                                 10.

00       Dipotassium hydrogen phosphate                                           2.00       EosinYellow                                                                         4.00         Methylene blue                                                                    0.065           Agar                                                                                     25.00         Final pH (at 25° C) 7.

2         Preparationof Medium        Suspended 36 gm ofdehydrated EMB in 1000 ml distilled water and sterilized by         autoclaving at 15psi pressure, 121° C for 20 minutes. The molten medium was cooled to        about50° C temperature and poured into sterile petriplates.         Use:For the isolation, cultivation, and differentiation of Gram-negativeenteric bacteria          based on lactose fermentation.Bacteria that ferment lactose, especially the coliform           bacterium Escherichia coli, appear as colonies with a green metallic sheen orblue-black to          brown color. Bacteria that do notferment lactose appear as colorless or transparent, light         purplecolonies.          Methyl Red (MR) reagent         Methyl red                                                                         0.1 gm                                               Ethyl alcohol (95 to 96 percent)                                      300.

0 ml         Dissolve the dye in alcohol and thenadd sufficient distilled water to make 500 ml.         Voges-Proskauer (VP) test reagents       1. Fivepercent ?- naphthol in absolute ethanol         ??. 40 percent KOH containing 0.3 percent creatine         Kovac’sreagent       Paradimethylaminobenzaldehyde                             50 gm         Pure amyl or isoamyl alcohol                                    75 ml         Concentrated pure hydrochloricacid                        25 ml         Dissolve the aldehyde in the alcoholby gentle warming in a water bath, cool and add the              acid. Protect from light and store at4° C temperature.

Kovac’s reagent A reagent used to              detect the presence of indole. Used inthe identification of bacteria.         Solutions for PCI method         Hydrationbuffer or storage buffer (1x TE)        TE                                                                                    per 100 ml         10 mM Tris-HCl (pH 7.

4)                      1 ml 1 M Tris-HCl pH 8.0 (0.1576gm in 1 ml)         1 mM EDTA (pH 8.

0)                         0.5 ml 0.5 M EDTA pH8.

0 (0.093 gm in 0.5 ml)           1x TE (Tris EDTA buffer) used as ahydration buffer as well as in storage of DNA.

        Lysis buffer                    per100 ml         1 M NaCl                                      2 ml         1M Tris HCL                              1 ml         0.5 M EDTA                                 0.2 ml         10% SDS                                      10 ml                Distilled water                              86.8 ml           10%SDS      Add 10 gram dissolved in 100 ml distillwater.

         0.5MEDTA        Add 18.62 gram in 100 ml distill water.         1NNaOH            Add 4 gram in 100 ml distill water.

         1M Tris HCL             Add 15.76 gram in distill water toreach 100 ml final volume, then autoclave.                                                                                                            


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