Materials and Methods

Transcription

1 Chapter-3 Materials and Methods 3.1 Isolation of Escherichia coli strain: 10 ml of sewage was collected and the residual solid matter was removed by filteration using Whatman No.1 filter paper. 0.1 ml of filtrate sewage was sprayed over the sterile plate of McConkey s agar and Eosin methylene blue agar aseptically. The experiment was performed in triplicate. The incubation of the plates were done at 37 0 C ± 2 0 C for 24 hours. Colonies showing typical characteristics of green metallic sheen on EMB agar and typical pink colour colonies on McConkey s agar were aseptically picked and purified by four-way streaking method on sterile Nutrient agar. The streak plate were incubated at 37 0 C ± 2 0 C for 24 hours and pure axenic culture were maintained on nutrient agar slant for further study (Cheesbrough M., et al., 1996) Composition of McConkey s - M008 Agar Bile Salts 1.50 Lactose Neutral Red 0.03 Peptic digest of animal tissue ph 7.0 ± 0.2 Proteose peptone 3.00 Sodium Chloride 5.00 Suspend gms in distilled water, heat to boil to dissolve the medium completely sterilized by 15 lbs pressure at C for 15 min. Mix well before pouring. (Hi-Media Manual, 2003) Composition of Eosin Methylene Blue (EMB) - M317 Agar Di-potassium Phosphate 2.00 Eosin Y 0.40 Lactose 5.00 Methylene Blue Peptic digest of animal tissue Sucrose 5.00 ph 7.0 ±

2 Suspend Gms in distilled water, heat to boil to dissolve the medium completely sterilized by autoclaving 15 lbs pressure at C for 15 min. Mix well before pouring. (Hi-Media Manual, 2003) Nutrient agar - M001 Gm/Lt Agar Beef Extract Peptone 5.00 ph 7.0 ± 0.2 Sodium Chloride 5.00 Suspend all ingredients in distilled water stir to dissolve and boil the medium to dissolve agar-agar completely and sterilization done by autoclaving on 15 lb at C for 15 min. (Hi-Media Manual, 2003) 3.2 Confirmation of Escherichia coli: In all 12 pure isolated colonies were obtained which were subjected to confirmation studies using standard conventional source tracking method Morphological Characterization : Gram Staining: All the 12 isolates were checked for gram reaction as per the method prescribed in (Cheesbrough M., et al., 1996). The smear was stained by crystal violate and fixed by using lugols iodine as mordant decolorization was performed by using acetone ethanol mixture and counter staining was done by using saffranin. Microscopic observations were done at 100x using Cidar oil as immersion fluid Composition of Gram staining: Crystal violet: Solution A: Ethyl alcohol Crystal violet Solution B: Ammonium oxalate 20 ml 3.00 gm 80 ml 0.8 gm 16

3 Mix Sol. A + Sol. B Lugols iodine: Iodine crystal Potassium iodide 1.0 gm 2.0 gm 300 ml Mix the iodine and potassium iodide in mortar and pestle until finally add in small amount and dissolve completely and make volume 100 ml in measuring cylinder. 90% Alcohol: 10 ml absolute alcohol mix with 90 ml distilled water. Saffranin : Saffranin 2.5 gm 95% Alcohol 100 ml 10 ml Scanning Electron Microscopy (SEM) : The isolates were subjected to Scanning Electron Microscopy at NBSS & LUP Nagpur and Shraddha Analytical Services Mumbai, for confirming the morphological changes due to stress responses (Klainer A. S, et al., 1970). Method: Preparation of bacteria for Scanning Electron Microscopy Growth in aerated liquid media at log phase- centrifuge at 1000 g for 30 minpellet fixed with 1% v/v gluteraldehyde for 30 min. fixed with 1% w/v osmium tetraoxide 12 to 20 hours dried pellets broken in to fragments gold/ palladium coating (60:40).- microscopy at 25 kv. (Kazunobu A and Akiko U, 1997) Materials For Scanning Electron Micrography: Glutaraldehyde 1% (v/v) Osmium Tetroxide 1% (w/v) Sodium Cacodylate buffer 0.15 M Ethanol % Cacodylate buffer ph 7.0 Amyl acetate 100% Gold/palladium (60 : 40) 17

4 Motility: The motility of 12 isolates were performed by hanging drop method as described in (Cheesbrough M., et al., 1996). Method The bacterial culture grown in a 10 ml sterile nutrient broth and incubation carried out at 37 0 C for 24 hrs. The tube taken without shaking and place a loopful culture on the surface of cover slip. The cover slip is placed on the cavity slide and fixed and inverted so that the drop of the culture becomes hanging. The hanging drop of the culture is then observed under 10x and 45x compound microscope and observed for boundary of the drop and mortality of the bacteria Materials for Motility: Cavity glass slide, Cover slip, Wax, Inoculating loop holder, 24 hour old bacterial culture, Compound Microscope Cultural characteristics: All 12 isolates regrown on Nutrient agar, EMB agar and McConkey s agar for typical cultural response as described earlier in Sugar Fermentation: The isolates are grown on typical carbohydrate fermentation media and acid/gas production was studied on Glucose, Arabinose, Lactose, Maltose, Mannose, Manitol, Sorbitol and Xylose as per method described in (Collee, et al., 1989). Material: Peptone 5 gm Sodium Chloride 5 gm Distilled Water Bromothymol blue 2 ml ph 7.00 ± 0.2 Glucose/Arabinose/Lactose/Maltose/Mannose/ 10 gm Manitol/Sorbitol and Xylose To make 1% sugar concentrations add each sugar into separate flask and prepare 1% sugar solution. Suspend all ingredient in distilled water stir to dissolve completely and sterilized by autoclaving on 15 lb at C for 15 min. (Hi-Media Manual, 2003) 18

5 3.2.4 IMViC Test: Indol Production, Methyl Red, Voges Praskaur Test, and Citrate utilization (Simmons) were performed as described in (Collee, et al., 1989) and the results were confirmed from Bergey s Manual. Indol Test: Tryptone water: Media: Sodium chloride Tryptone 10.0 gm Reagent: kovacs reagent- Para-dimethyl amino benzaldehyde 2.0 gm Isoamyl alcohol 30 ml Conc. HCl 10 ml Methyl Red test: Glucose Phosphate Broth: Media: Di-potassium hydrogen phosphate Glucose Peptone ph 7.4 Reagent: Methyl red Absolute alcohol 28 ml 22 ml. Dissolve the stain in alcohol completely and then add distilled water. Voges proskauer: Glucose Phosphate Broth: Media: Di-potassium hydrogen phosphate Glucose Peptone ph 7.4 Reagent: Barrits Reagent: Barrits A: KOH 40 gm 100 ml Barrits B : 5% alpha- napthol Absolute alcohol 100 ml Mix Barrits A and B to dissolve 19

6 Citrate Utilization Test: Simmons citrate Agar: Media: Agar 20.0 gm Ammonium Dihydrogen Phosphate Bromothymol Blue 0.02 % Distilled Water Potassium di-hydrogen phosphate 10.0 gm Sodium chloride Sodium Citrate 20.0 gm ph 6.8 ± Enzyme Studies: Hydrogen sulphide production, Urea hydrolysis, Phenylamine deaminase and Lysine decarboxylase studies conducted on specific media obtained from Hi-Media. Lipase, ONPG and oxidation fermentation test were performed according to standard method described in (Collee, et al., 1989) and results were confirmed from Bergey s Manual 9 th Edition. Materials: composition. 1) H 2 S Production Media-Triple Sugar Iron Agar- M021 Agar Beef extract 3.00 Casein enzymic hydrolysate Dextrose 1.00 Ferrous Sulphate 0.20 Lactose Peptic digest of animal tissue Phenol Red Sodium Chloride 5.00 Sodium Thiosulphate 0.30 Sucrose Yeast extract 3.00 ph 7.4 ± 0.2 Suspend 65 Gms in distilled water. Heat to boil to dissolve the medium completely, mixed well and distribute in the tubes. Sterilized the media on 15lb at C for 15 min. to cool and keep the tubes in tilting position to make slants. (Hi-Media Manual 2003) 20

7 2) Urea hydrolysis Media-Triple Sugar Iron Agar- M111 Dipotassium Phosphate 9.50 Monopotassium Phosphate 9.10 Phenol Red 0.01 Yeast extract 0.10 ph 7.4 ± 0.2 Suspend 18.7 gms in 950 ml distilled water.. Sterilized by autoclave on 15lb at C for 15 min., cool to 55 0 C. Aseptically add 50 ml of sterile 40% urea solution (ED048). Mix well, distribute the medium into the plates and solidify. (Hi-Media Manual 2003) 3) Phenylalanine deaminase Media-Phenyl Alanin Agar-M281 Agar DL-Phenylalanine phosphate Sodium chloride Yeast extract Disodium Final ph 7.3±0.2 Add 26 grams in de-ionized/distilled water. Heat to boil till the contents are completely dissolved, distribute into tubes and sterilize at15 lb pressure using autoclaving for 15 minutes. Allow the tube to cool and place in slanting position. (Hi-Media Manual, 2003) 4) Lysine decarboxylase Media- Lysine decarboxylase broth M376 Bromocresol Purple 0.02 Dextrose 1.00 L-lysine hydrochloride 5.00 Peptic digest of animal tissue 5.00 Yeast extract 3.00 ph 6.8 ± 0.2 Suspend 14 Gms in distilled water. Heat to boil to dissolve the medium completely, mixed well and distribute in the plates. Sterilized the media on 15lb at C for 15 min. to cool to solidify the agar. (Hi-Media Manual 2003) 21

8 5) ONPG Media ONPG Discs DD008 Materials: Take a sterile test tube and placed one ONPG disc. 0.1 ml of sterile physiological saline is added. The colony under test is picked up and emulsified in physiological saline in the tube containing disc. Incubation is done at 35 to 38 o c. observe the tube for 6 hours at an interval of one hour. For late lactose fermentation incubate the tubes for 24 hours. (Hi-Media Manual, 2003) 6) Lipase Medium: Peptone 5.00 Beef extract 3.00 Sodium Chloride 5.00 Agar ph 7.0 ± 0.2 Add 50 ml Egg Yolk Emulsion in 1 lt. sterile nutrient agar at 40 0 C temperatures, and pour in to petriplates. Cool the medium to solidify. Spot inoculate the culture at the center of the plate and incubate at 37 0 C ± 2 0 C for 24 hours. Observe zone of hydrolysis (Hi-Media Manual, 2003) 7) Oxidation / Fermentation ( O F) Test Medium: Basal Medium without Carbohydrate: Peptone 2.00 K 2 HPO Sodium Chloride 5.00 Bromothymol Blue 0.03 Agar ph 7.0 ± 0.2 Heat with agitation to dissolve agar. Dispense 3 ml portion in to 13x 100 mm tube. Autoclave for 15 min at C cool to 50 0 C ph 7.1 (Frances Pouch, 2001). 22

9 Carbohydrate Stock solution: Dissolve 10 gm carbohydrate in 90 ml distilled water sterilized by filtration through 0.22 micrometer membrane filter. Add 0.3 ml stock solution to 2.7 ml base in tubes. Mix gently and cool at room temperature. Inoculate tubes in duplicate. Layer one tube with sterile mineral oil. Incubate for 48 hours at 35 0 C. (Frances Pouch et. al., 2001) 3.3 Confirmation of Standard Escherichia coli: Escherichia coli culture was procured from National Chemical Laboratory (NCL), Pune with Code No.NCIM 2065 which was ATCC culture hereafter referred to as Escherichia coli ATCC This culture was maintained after repeated subculture at 4 0 C. The culture was confirmed by gram staining, motility, growth on EMB agar and McConkey s agar, IMViC, and lactose fermentation. The culture shall be maintained at 4 C with repeated sub-culture every three months. The standard procedure shall be used as per the method prescribed (Lapase S. P. et al., 1970). 3.4 Stress response studies on first transition element on Escherichia coli ATCC 8739 and Escherichia coli isolate in solid state. Method: Out of 10 transition element eight elements were selected for stress response studies namely, Vanadium pentoxide, Chromium nitrate, Manganous acetate, Ferric oxide, Cobalt chloride, Nickel nitrate, Cupric sulfate, and Zinc sulfate (Atomic No. 23 to 30). All the eight elements were prepared in six different concentrations by serial dilution and concentrations of salt understudy were 0.2µgm/ml, 2.0µgm/ml, 20µgm/ml, 200µgm/ml, 2x10 3 µgm/ml and 2x10 4 µgm/ml. Sterile nutrient agar plates were prepared in triplicate. 24 hours cultures of Escherichia coli ATCC 8739 and Escherichia coli isolate were grown on nutrient growth. 1.0ml of these cultures were poured into molten agar and poured into sterile plates for preparation of culture plates. Well was cutout at the centre using a sterile well cutter of 0.5 cm in diameter. The well was sufficient to hold 0.5ml of salt 23

10 solution. 0.5ml of sterile salt solution of each concentration were added per well per plate. The plates were incubated at 37 0 C ± 2 0 C for 24 hours in straight position. The zone of inhibition was measured using Hi-Media Zone Measuring Scale. This procedure was followed according to the method prescribed in (Benson H. J. 1990). 3.5 Stress response studies on Escherichia coli ATCC 8739, and Method: Escherichia coli isolate in aqueous state. Escherichia coli being a non fastidious organism can profusely grow on nutrient growth hence this medium was selected for aqueous phase exposure studies. Both reference strain and test organism were grown in 100 ml nutrient broth medium taken in 250 ml flask. The flasks were incubated at 37 0 C ± 2 0 C, for 24 hours. Every three hours an aliquot of 1 ml was aseptically removed and Standard Plate Count (SPC) was performed to find out the CFU/ml. The CFU/ml at 6 hours growth was standardized by repeated experimentation. The optical density of the growth of standard CFU at 6 hours was measured at 600 nanometers. Sets of freshly prepared nutrient broth were prepared and inoculated with 6 hours growth standard culture as per MacFarland standard. Separated tubes were then exposed to the metal salts from vanadium to zinc (At.No.23 to 30) with concentration 0.2µgm/ml, 2µgm/ml, 20µgm/ml, 200µgm/ml, 2x10 3 µgm/ml and 2x10 4 µgm/ml. The culture flask was then incubated at 37 0 C ± 2 0 C for 6 hours. 5 ml of aliquot of culture were removed aseptically and optical density was measured at 600 nm. The percent inhibition was calculated as follows: O.D. of Standard O.D. of Test Percent inhibition = x 100 O.D. of Standard 3.6 Variation in enzyme profile due to exposure to first transition elements stress: Method: Test organism and Reference strain which showed either stress response or tolerance were picked up from each metal exposure studies and were subjected to enzyme production studies. 24

11 3.6.1 Production of Enzyme Tryptophanase: Escherichia coli are known to produce enzyme Tryptophanase and hence are referred to as Indol positive organism. Sterile tryptone broth was prepared and 10 ml of media were dispersed in a series of cotton plug test tubes. Each tube was inoculated with test organism obtained from exposure studies showing either stress or tolerances. Experiments are performed separately for Reference as well as Test organism. The tubes were incubated at 37 0 C ± 2 0 C for 24 hours. 5 ml aliquot was removed and the production of Indol was confirmed by adding Kovac s reagent (Collee, et al., 1989). The cherry red colour of Indol complex was taken out as 0.5ml of this complex was diluted with 1.5ml of Amyl alcohol. The optical density (O.D.) of complex was measured at 450 nm (Collee, et al., 1989). The percent change in the enzyme production was calculated as: O.D. of Unexposed Culture O.D. of Exposed Culture Percent change of = x 100 production of Tryptophanase O.D. of Unexposed Culture Material: Tryptone water: Media: Tryptone NaCl Distilled Water Reagent: kovacs reagent- Para-dimethyl amino benzaldehyde Isoamyl alcohol Conc Hcl (Hi-Media manual 2003) 10.0 gm 2.0 gm 30 ml 10 ml Production of enzyme Amylase: Method: 1% starch agar plates were prepared and they were mixed with 1 ml of metal salts containing 0.2µgm/ml, 2.0 µgm/ml 20µgm/ml, 200µgm/ml, 2x10 3 µgm/ml, 2x10 4 µgm/ml of the respective metal salts. The plates were solidified, test organisms as well as reference strains were separately spot inoculated at the centre of the plate with culture diameter of 2 mm. The plates were incubated at 37 0 C ± 2 0 C for 24 hours. The plates were then flooded with lugols iodine and zone of hydrolysis was measured in mm (Collee, et al., 1989). 25

12 The percent change of hydrolysis was calculated as: Zone of Hydrolysis of Zone of Hydrolysis of Percent change of Unexposed Culture Exposed Culture hydrolysis of starch = x 100 Zone of Hydrolysis of Unexposed Culture Material: 1% Starch Agar: Media: Beef extract 3.0 gm Peptone Sodium chloride ph 6.8 ± 0.2 Starch Powder 10 gms (Hi-Media Manual 2003) Production of enzyme protease: 1% Bovine serum Albumin Fraction 5 was mixed with molten Nutrient agar and sterile plates were prepared. The plates were spot innouculated with 2 mm diameter and incubated at 37o C +- 20c for 24 hours. The zone of hydrolysis was measures and the percentage change in protease was calculated as- Zone of Hydrolysis of Zone of Hydrolysis of Percent change of Unexposed Culture Exposed Culture Production of Protease = x 100 Zone of Hydrolysis of Unexposed Culture Material: 1% Bovine Serum Albumin (BSA) Agar: Media: Beef extract 3.0 gm Peptone Sodium chloride ph 6.8 ± 0.2 Bovine serum albumin 1% (Hi-Media Manual 2003) 26

13 3.7 Variation in RNA profile due to exposure to First Transition Elements Stress: Method: RNA was isolated from overnight grown Escherichia coli in LB broth and was estimated by orcinol method. The total amount of RNA was represented in microgram per ml. After digestion of DNA the total RNA was separated by agarose gel electrophoresis and the bands of RNA were visualized using ethidium bromide. The control and metal exposed strains of E. coli were compared for estimating the total effect. Material: Orcinol Method: 1. RNA solutions 0.2 mg/ml 2. Orcinol Reagent (Dissolve 1 gm of Ferric chloride (FeCl 3.6H 2 O) in 1 liter of concentrated HCl and add 35 ml of 6% w/v Orcinol in alcohol. (David T. Plummer 2001) Isolation and Electrophoresis: 1. Phenol solution 900 gm/lt 2. Potassium acetate (200 gm/lt, ph 5) 3. Absolute Ethanol 4. Diethyl ether Electrophoresis: 1. Potassium hydroxide 2. Perchloric acid (200 gm/lt) 3. Citrate Buffer 0.02 mol/lt 4. ph HCl (0.1 mol/lt) 6. Agaros Gel 1% Luria Bertani (LB) Medium: 1. Deionised water 1000ml 2. Bactotryptone 10 gm 3. BactoYeast 5 gm 4. Sodium hydroxide 5 mol. 5. HCl 5 mol. (David T. Plummer 2001) 27

14 Protein by Folin-Lowry method: Materials: 1. Alkaline Sodium carbonate solution (20gm /lt Na2CO3in 0.1 mol/lt NaOH- Solution A 2. Copper sulphate, Sodium potassium Tartarate solution 5 gm/lt CuSO 4. 5H 2 O in 10 gm/lt Na, K-Tartarate - Solution B 3. Alkaline solution 50 ml Solution A + 1 ml Solution B 4. Standard Phenol Reagent - 5. Standard protein, Bovine Serum Albumin 0.2 mg/ml (P. No.159 An Introduction to practical biochemistry, David T. Plummer 3 rd Ed. Tata McGraw Hill 2001) 3.8 Statistical Analysis: For a thorough understanding of the data, the results were subjected to various statistical analysis including unpaired student t-test, One way ANOVA with multiple choice and comparison. All the analysis were done using the software of prism pad Version V. 28