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.
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.
