3. Materials and Methods 3.

1 Collection of Soil Samples for Bacterial Strains Isolation The soil samples containing the bacteria were collected from different areas of Tadaepalli and Mangalagiri, Guntur District of Andhra Pradesh where there was an accumulation of the petroleum spills for Lead deposits and where as for Mercury degradation samples were collected from Seashore of Suryalanka, Baptla, Guntur District, Andhra Pradesh. The soil sample was collected from a depth of 15 inches depth in to the soil where there was high moisture content, because the Bacteria need moisture content for growth. The soil sample collected was dispensed in the sterile bags and sealed and the sample was brought to the laboratory.

3.2 Screening of Lead and Mercury Degrading Bacteria Soil samples were collected from the sites where lead and Mercury effluents were discharged. Different media like Nutrient Agar, Lauria Bertani agar plates containing Lead and Mercury to a final concentration of 5.0ppm and 7.5ppm respectively. Ten fold serial dilutions of the soil samples were made in distilled water and 0.1ml of 10-5 to 10-9 dilutions were plated on different media. The plates were incubated at 37C for 24 hours and observed for clearance of degradation surrounding the colonies after incubation. The colonies, which cleared the degradation, were transferred to similar agar plates containing 5.0 ppm of Pbcl2 for single colony isolation of Lead degrading organism. Where as in another set of plates 7.5 ppm concentration of HgCl2 is used for Single colony isolation of Mercury degrading organism Isolation. The isolated colonies were streaked on Nutrient

agar slants for growth and maintenance. Stock cultures were sub cultured monthly and stored at 4C. The cultures were confirmed based on morphology by Grams staining. Media and culture conditions The medium used for the maintenance of the isolated fungal strain was Potato Dextrose agar medium with the following ingredients: Composition Ingredients Peptone Beaf Extract Sodium Chloride Agar Distilled water Final pH Amount 5g 3g 3g 2% 1000ml 7.2-+0.2

a. The above ingredients were weighed and dissolved in distilled water in a conical flask. b. The pH of the medium was adjusted by adding acid or alkali.
c. The conical flask containing the medium was autoclaved at 1210c at 15 lbs pressure

for 15 min. d. 15ml of the medium was dispensed in to sterile petriplates and allowed to solidify.

The Bacterial cultures were streaked on Nutrient Agar slants. Stock cultures were sub cultured monthly and stored at 4C.

3.3 Identification of the Bacteria The isolated bacterium was identified using different staining techniques, different biochemical and molecular techniques. 3.3.1 Simple Staining Bacteria are very small and transparent when observed with a wet mount preparation. In order to observe their cell characteristics, they need to be stained (Dyed). This method consists of preparing a smear that is air dried and heat fixed and adding a stain to the bacteria on the slide. The simple stain consists of one dye. The dye adheres to the cell wall and colors the cell making it easier to see. Basic stains, such as methylene blue, Gram saffranin or Gram crystal violet are used for staining the most bacteria. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged. Since the surface of most bacterial cells is negatively charged, these positively charged stains adhere readily to the cell surface. Materials Heat-fixed bacterial smears. Methylene blue, Crystal violet, or Saffranin to act as simple stain. Ppaper towels.

Microscope. Procedure A small drop of water was placed on a slide with a toothpick making the culture to be in contact with water and air dried. The dried slide was placed on the staining rack and flooded for 1 minute with the stain. The slide was rinsed with tap water, tilting the slide slightly to washout all the stain from the slide by gently tapping. The slide was placed on the bibulous paper and blot dried. The slide was examined under the microscope and the results were recorded.

3.3.2 Gram Staining Several different kinds of bacteria will be examined by gram staining, a method developed by Christian Gram in 1884 for categorizing bacteria on the basis of differences in cell wall structure. Gram positive (+) bacteria stain a dark purple color, while gram negative (-) bacteria stain a light red.

Microscopy reveals at least two different types of cell wall structure: single and double. The walls of gram +ve bacteria consist of a single, thick, continuous layer, whereas those of gram ve bacteria consist of at least two readily

distinguishable layers, each considerably thinner than the wall of a gram +ve bacterium. In addition, chemical analyses reveal substantial differences in the chemical composition of the two kinds of cell walls. In preparing a gram stain, the first step stains both kinds of cells, gram +ve and gram ve, with a deep purple dye complex. The next steps expose the cells to a decolorizing agent such as alcohol and then to the counterstaining red dye Saffranin O. Since the cell wall of the gram +ve bacterium constitutes a barrier to the decolorizing agent, the gram +ve bacterium remains purple, while the gram -ve bacterium is decolorized and appears light red due to the color of the red Materials and Reagents 24 h old culture Inoculating loops Blotting papers Bunsen burner and Microscope Crystal Violet (Huckers) Solution A 2g Crystal Violet (90% dye content)

 20 ml

Ethyl

alcohol

(95%)

Solution B 0.8 g 80 ml Distilled water Ammonium Oxalate

Dissolve the crystal Violet in ethy alcohol an dthe ammonium oxalate in distilled water, Mix solutions A and B Grams Iodine Iodine Potassium iodide Distilled water 1g 2g 300 ml

Dissolve iodine and potassium iodide in distilled water

Ethyl alcohol (95%)

Ethyl alcohol (100%) Distilled water Safranin (2.55 solution in 95 % ethyl alcohol) Distilled water

95 ml 5 ml 10 ml 100 ml

Procedure for gram staining A small drop of water was placed on a slide with a

toothpick making the culture to be in contact with water and air dried. The dry slide was passed through a Bunsen burner flame

for 2 to 3 minutes to heat fix the smear. The slide was placed on a staining rack and the slide was

flooded with crystal violet for 2 minutes. The slide was rinsed with water from a squirt bottle. The slide was flooded with grams iodine for 1 minute and

rinsed with water. Then the slide was subjected to decolonization step with

95% ethanol by adding 20 drops of 95% ethanol to the slide and the slide was left for 10 seconds and then rinsed with water from a

squirt bottle. Only gram + bacterial will retain the deep purple dye. The gram bacteria will be colorless at thwas point. Then the slide was counterstained with Saffranin for 60

seconds. This was carried out to visualize materials that have not been stained by the deep purple crystal violet. Then the slide was rinsed with water from a squirt bottle

and blot dried. The slide was observed under the microscope (Olympus

Inverted microscope) first with low power objective and the slide was placed under immersion oil directly on the base of the microscope and it was examined with an oil immersion objective. 3.4 Biochemical Tests

3.4 Biochemical Tests 3. 4.1 Indole production test Tryptophan an essential amino acid is oxidized by some bacteria by the tryptophanase resulting in the formation of indole, pyruvic acid and ammonia. The indole test is performed by inoculating a bacterium into tryptone broth, the indole produced during the reaction is detected by adding Kovacs reagent (Dimethyl amino benzaldehyde) which produces a cherry-red reagent layer. Requirements Nutrient broth cultures

Tubes containing 1% Tryptone broth, 5 ml/tube Kovacs reagent Dropper bottle 1 ml pipette Bunsen burner Inoculating needle

Preparation of tryptone broth (pH 7.0) Tryptone Calcium chloride Sodium chloride Distilled water 10 g 0.01-0.03 M 5g 1000 ml

Procedure Tryptone broth was prepared as per the composition. Tryptone broth tubes were inoculated with the cultures and keep

one tube was kept as control.

The inoculated tubes were incubated at 350C for 48 h. After 48 h of incubation, I ml of Kovacs reagent was added to

each tube including control. Shake the tubes to stand to permit the reagent to come to the top.

3.4.2 Methyl red and Voges proskauer tests The methyl red test and VP test are used to distinguish between bacteria that produce large amount of acid and those that produce the neutral product acetoin as an end product. Both these are performed simultaneously because they are physiologically related and are performed on the same medium MR_VP broth. In these tests, if an organism produces large amount of organic acids (end products) from glucose the medium will remain red (a positive test) after the addition of methyl red a pH indicator (pH <4.4). MRVP test is of value in the separation of E.coli and Enterobacter aerogenes which appear virtually identical except for certain physiological differences that are used as indicators of the sanitary quality of water, foods, food production and eating establishments. Requirements Nutrient broth cultures V. P broth tubes 5 ml/tube Methyl red PH indicator V. P reagent 1 (naphthol solution) V. P reagent 2 (40% potassium hydroxide) Clean empty test tubes

Bunsen burner Inoculating loop

Preparation of MRVP broth (pH 6.9) tubes Peptone Dextrose/Glucose Potassium phosphate Distilled water 5g 10000 ml 7g 5g

Procedure 5 ml broth was poured in each test tube and sterilized by autoclave

at 15 lbs pressure for 15 minutes. MRVP tubes were inoculated with test cultures and one tube was

kept as control.

The inoculated tubes were incubated at 350C for 48 h. 5 drops of methyl red indicator was added to the tube of each set. 12 drops of V-P reagent was added to the test tubes. The tubes were shaken gently and the caps were off to expose the

media to oxygen.

The reaction was allowed to complete for 15-30 minutes.

3.4.3 Gelatin Hydrolysis (Production of Gelatinase) Gelatin is a protein produced by hydrolysis of collagen a major component of the connective tissue and tendons in humans and other animals, Hydrolysis of gelatin is brought about by microorganisms capable of producing proteolytic exoenzyme known as gelatinase, which acts to hydrolyze this protein to amino acids, Hydrolysis of gelatin can be identified by growing the microorganisms in the nutrient gelatin. If the degradation of gelatin occurs in the medium by an exoenzyme produced by the bacteria , it can be detected by lequification by flooding the plates with the protein precipitating material (By adding the gelatin plates with mercuric chloride solution and observing for the clearing around the line of growth). Materials required 24 h culture Gelatin agar medium (Hi Media) Mercuric chloride solution Sterile petri dishes Inoculating loops

Procedure

The gelatin agar medium was prepared according to standard

protocol and sterilized at 121C for 15 minutes and cooled to 45 to 50C. The sterilized medium was poured in to sterile petri dishes and solidify.

allowed to plates.

The culture was inoculated by streaking on to the gelatin agar

The plates were incubated at 37 C for 4 to 7 days. The plates after incubation period were flooded with mercuric

chloride solution and the plates were allowed to stand for 5 to 10 minutes. 3.4.4 Starch Hydrolysis Amylase is an exoenzyme produced by the bacteria the hydrolysis starch a polysaccharide, the ability of the bacteria to produce amylase is determined by adding a indicator iodine solution starch in the presence of iodine produces dark blue coloration of the medium and an yellow zone around the colony which indicate the amylolytic activity Requirements 24 h old culture. Starch Agar Medium (Hi Media). Grams iodine solution. Sterile petri dishes. Dropper.

Inoculating loop.

Procedure The starch agar medium was prepared according standard protocol

and sterilized at 121C for 15 minutes and the medium was cooled to 45C. The sterilized medium was poured in to sterile petri dishes and

allowed to solidify. Each of the starch plate was marked with the name of the organism The culture was inoculated by streaking on to the starch agar plates The plates were incubated at 37 C for 48 hours in inverted

position. The incubated plates were flooded with iodine solution with a

dropper for 30 seconds. The excess iodine solution was poured off. The plates were examined for the colour change in the medium

after the addition of the iodine solution which indicates the hydrolysis of starch. 3.4.5 Oxidase Production The production of oxidase is one of the most significant tests we have for differentiating certain groups of bacteria. For example, all the Enterobacteriaceae are oxidase-negative and most species of Pseudomonas are oxidase-positive.

Another important group, the Neisseria, are oxidase producers.

This test: The

first method utilizes the entire TSA plate; the second method is less demanding in that only a loopful of organisms from the plate is used. Both methods are equally reliable. Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (An iron containing haeme protein). These both catalyse the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, N, N, N, N-tetra-methyl-p-phenylenediamine dihydrochloride acts as an artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the coloured compound indophenol blue. The cytochrome system is usually only present in aerobic organisms which are capable of utilizing oxygen as the final hydrogen receptor. The end product of this metabolism is either water or hydrogen peroxide (Broken down by catalase).

Requirements 24 h old culture. Nutrient Agar Plates N, N, N, N-tetra-methyl-p-phenylenediamine dihydrochloride Bunsen burner. Inoculating loop.

The medium Nutrient Agar (pH 7) plates was prepared according

to the stranded procedure and the medium was sterilized at 121C for 15 minutes.

The Nutrient Agar plates were inoculated with the culture. The inoculated plates were incubated at 35C for 24 to 48 h. Pour a few drops of a freshly prepared solution of 1% N, N, N, N-

tetra-methyl-p-phenylenediamine dihydrochloride over the colony to be tested. Colonies which change to a deep blue colour indicate a positive reaction.

3.4.6 Catalase Activity During aerobic process that take place in the presence of oxygen in the bacterial cell, H2O2 is produced which is lethal to the cell. The enzyme catalase is also produced by the bacteria that produced H2O2. The enzyme catalase breaks down the hydrogen peroxide to water and oxygen and helps the bacteria to survive. Catalase 2 H2O2 -------------- 2H2O + O2

This test was performed by adding H2O2 to Tripticase soya agar plates. The addition of H2O2 results in formation of the bubbles which indicate the release of the oxygen which mean the test is positive Requirements 24 h old culture. Trypticase soya agar plates. Hydrogen peroxide (3%). Bunsen burner. Inoculating loop. Trypticase Soya Agar composition Trypticase Phytone Sodium Chloride Agar 15 g (Animal peptone) 5 g ( Soyapeptone) 5g 15 g

Make upto 1000 ml with Distilled water Procedure

The medium Trypticase soya agar (pH 7.3) plates was prepared

according to the stranded procedure and the medium was sterilized at 121C for 15 minutes. The Trypticase soya agar plates were inoculated with the culture. The inoculated plates were incubated at 35C for 24 to 48 h. The inoculated plates were observed for the presence or absence of

the gas bubble.

The formation of the gas bubbles on addition of the H2O2 indicate

the release of free oxygen which indicate the test is positive for production of catalse by the bacteria

Utilization of macro-nutrients by Lead and Mercury Degrading isolates The basal medium contained

Glucose, 10 g NaN03, 2 g MgS04.7H20, 0.2 g CaCl2, 0.3 g Thiamine hydrochloride, 0.5 mg Distilled water-1000 ml

Procedure

The ingredients of basal medium were weighed and dissolved in distilled water in a conical flask.

The pH of the medium was adjusted to 5.5 by adding either acid or alkali. Then the conical flask containing medium was autoclaved at 15 lbs pressure for 15 minutes at 121OC

Antibiotic (Streptomycin) was added to the medium after cooling and mixed gently. Then the medium was inoculated with the loop of Pleurotus isolates. Each macro-element to be used, one having all the macro -nutrients and the other having none.

Then the flasks were kept at room temperature for growth observation.

3.10. Effect of trace elements on the degradation of Lead and Mercury isolates The ingredients of basal medium were weighed and dissolved in distilled water in a conical flask.

The pH of the medium was adjusted to 5.5 by adding either acid or alkali. Then the conical flask containing medium was autoclaved at 15 lbs pressure for 15 minutes at 121OC

Antibiotic (Streptomycin) was added to the medium after cooling and mixed gently. Then the medium was inoculated with the loop of Pleurotus isolates. Five trace elements (copper, iron, manganese, cobalt and zinc in their sulphate form), were added separately to the basal medium at the rate of 10 mg/1000 cm3.

Two sets of controls were also prepared. The first consisted of all the trace elements while the second set contained basal medium without any micro-element.

Then the flasks were kept at room temperature for growth observation. Isolation of DNA from Mercury and Lead Degrading Isolates The DNA from Mercury and Lead degrading isolates were prepared as per the protocol

1.5ml of overnight culture of each isolate was taken into different 1.5ml centrifuge tubes,

Centrifuged at 8000rpm for 5min. Supernatant was discarded and briefly vortex pellet to dislodge. 400l of Lysis buffer [1.5M NaCl, 10mM Tris-HCl (pH8.0), 5% SDS] was added to the

dislodge pellet and incubated in water bath for 15min at 65C. The solution was cooled to room temperature and 130l of potassium acetate (pH5.2) was

added and mixed. The solution was incubated for 5 minutes in room temperature.

The sample was centrifuged at 10000rpm for 10min.Clear supernatant was transferred into

fresh 1.5ml tube. Equal volume of isopropanol was added, gently inverted for 5 times and incubated 10min in room temperature.

The sample was centrifuged at 10000rpm for 10min. Supernatant was discarded and pellet

is washed with 75% ethanol.

The pellet was dried in room temperature for another 15 minutes. Pellet was dissolved in

100l of TE buffer. To remove RNA from the preparation, 10l of RNase A (10mg/ml) was added and incubated at 37C for 1 hour. 3.12.4 Purification of Isolates DNA Solutions

RNase A: RNase A of 10 mg/ml was dissolved in 10 mM Tris-HCl (pH 7.5), 15 mM NaCl. The contents were heated at 100oC for 15 minutes and allowed to cool slowly at room temperature. Dispensed into aliquot and stored at 20oC. Procedure

5 l/ml RNase solution (the amount of RNase depends upon the RNA contamination)
was added to the crude DNA and incubated at 37oC for 45 minutes.

To the above, 1 ml of TE saturated phenol was added, mixed the contents thoroughly and
then centrifuged at 15,000 x g at 4oC. The upper aqueous phase was transferred to a fresh tube and added with equal volume of a mixture of phenol: chloroform: isoamylalcohol (25:24:1) to the solution and mixed thoroughly without vortexing.

The contents were centrifuged at 15,000 x g at 4oC for 5 minutes and transferred the
upper aqueous phase to a fresh tube.

And to the contents, an equal volume of Chloroform: Isoamylalcohol (24:1) was added
and centrifuge at 15,000 x g at 4oC for minutes. This step is repeated until no precipitate is seen.

To the 1/10th volume (of the aqueous phase) of 3M Sodium acetate (pH 5.2) was added,
mixed the contents it and then added with twice its volume of chilled absolute ethanol. Mixed the contents thoroughly by inverting the tubes and incubate at 20oC for one hour to get precipitate of the DNA.

 The contents were centrifuged at 15,000 x g for 5 minutes at 4oC. The DNA was
collected discarding the supernatant and the pellet was air dried to remove ethanol and dissolved in appropriate volume of TE buffer. Amplification of the 16s rRNA Gene of the Bacterial Chromosome The polymerase chain reaction is an enzyme catalyzed biochemical reaction in which small amount of the specific DNA sequences are amplified into large amounts of linear double stranded DNA (Millis, 1990). PCR is used to amplify the DNA sequence in between two known sequences. The 16s rRNA gene of the bacteria was carried out in our laboratory in the Thermocycler (Eppendorf). In PCR the specific primers (Forward and reverse primers) complementary to the known sequences were added along with the master mix (HELINI Biomolecules, Chennai) to the DNA and the mixture was placed in a thermocycler heated at 94C for denaturation of the DNA. The mixture is then allowed to cool enabling the primers to anneal to the complementary sequences. A heat stable DNA polymerase was used to make the copies of the DNA form the original DNA sequence About 30 cycles of the DNA amplification was performed that resulted in a very large amplification of the DNA . The 1542 bp rRNA gene was amplified using two primers. Two primers annealing at the 5' and 3' end of the 16S rDNA were (Forward Primer) 5'

-GAGTTTGATCCTGGCTCAG-3' (positions 927 [Escherichia coli 16S rDNA numbering]) and (Reverse primer) 5'-AGAAA GGAGG TGATC CAGCC-3' (positions 15421525 [E. coli 16S rDNA numbering]) were used. The master mix containing 10X Taq buffer, 10 mM dNTPs, 25 mM of MgCl2, 1 U of Taq DNA polymerase,1.5 l of

forward primer, 1.5 l of Reverse primer, 100 ng of Genomic DNA and PCR grade molecular water to make the final volume to 20 l was used. Taq DNA polymerase initiates the replication of DNA fragments by using nucleotide base from dNTP mixture (A,T,G,C). Procedure 3.12.7.1 Setting up the PCR reaction A fresh master mix tube which contains the dNTP and Taq DNa polymerase was taken and the following components are added

Master Mix vial Forward Primer Reverse primer Template DNA Nuclease free water -------

10 l 1.5 l 1.5 l 2 l 5 l

20 l

(Master Mix contains 1U Taq DNA polymerase, 10X Taq buffer, 10 mM dNTPs and PCR grade water. Primer dye mix contains 10 uM forward primer, 10 uM reverse primer and PCR compatible dye with glycerol). After addition of all the components the PCR tube is gently spun down in centrifuge briefly and is placed in the Thermal cycler. The thermal cycler was programmed as follows.

Program 1 (one cycle) (Initial denaturation) 94C for 2 minutes Program 2 (30 cycles) (Amplification) Step one (denaturation) 94C for 45 seconds Step two (annealing) 56C for 1 minute Step three (extension) 72C for 1 minute 30 seconds Program 3 (one cycle - final extension) 72C for 5 minutes, then hold at 4C The programmed temperatures of the PCR reaction is represented in the diagram shown below

3.12.7.2 Agarose Gel Electrophoresis Gel electrophoresis is a technique used for the separation of nucleic acid and protein, where electrically charged molecules migrate at a rate proportional to their charge in mass ration when place under an electric field. It is undoubtedly the routine technique to monitor the success of the nucleic acid isolation procedures and analyze enzymatic manipulations such as restriction enzyme digestion. About 1.8 g of agarose was weighted and taken into 100 ml reagent bottle. To it 100 ml of 1X TBE buffer was added and it was heated in a microwave oven till all agarose gets melted up. The agarose solution was then poured in to gel casting unit assembled with appropriate comb and it was allowed to polymerize. After the polymerization the comb was removed and the gel was placed in an electrophoretic tank consisting of 1X TBE buffer. About 12 l of the 16s rRNA amplified DNA was mixed with 2 l of the gel loading dye (Bromophenol blue 6x) and it was loaded in 1.8 % agarose gel The gel was electrophoresed at 90 volts for about 30 minutes and it was observed in a gel documentation system.

3.12.7.3 Eluting DNA from agarose gel fragments Ethidium bromide stained agarose gel was visualized under a transilluminator on low setting. The fragment of interest was excised with a clean razor blade. After removing the excess liquid, the agarose fragment was placed in the spin column. The tube was centrifuged at 5500 rpm for not more than 45 seconds for the elution of DNA. The eluent was checked using a transilluminator for the presence of ethidium bromide stained DNA. The eluted DNA was used directly in enzymatic reactions. This DNA fraction was now subjected for sequencing.

3.12.7.4 Sequencing of Purified Product The purified product was subjected for DNA sequencing and it was carried out at..

3.12.7.5 Sequence match by NCBI Blast analysis The sequence of the purified product so obtained was subjected to BLAST analysis by using NCBI database to identify whether the obtained sequence was coding for the related organisms or not. Confirmation can be brought down by the highest percentage of similarity with the known sequences in the database.