EMB plates incubated overnight at 37° C. Metallic

EMB plates incubated overnight at 37° C. Metallic green sheen colonies in EMB plates were considered as presumptive E. coli and gloppy black colonies with a green metallic K.Pneumoniae .Gram-negative bacilli, motile strains with Methyl red and Indole positive (+), and Voges – Proskauer and Citrate utilization (-) and also which can ferment most of the sugar were presumptively considered as E. coli and  Methyl red and Indole negative (-), and Voges – Proskauer and Citrate utilization (+) K. pneumonia. All E. coli & K. pneumonia isolates were sub cultured on Luria agar & stored in stab culture as well as 20% glycerol stock at -80° C for long time storage.

3.2.9 Maintenance of Cultures

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3.2.9.1 Glycerol stock

Those colonies which were considered as presumptive E. coli & K. Pneumonia were transferred to Luria broth which is also referred to as Lysogeny broth (nutrient-rich medium that allows rapid and robust growth of bacteria) (Hi Media, Mumbai, India) and incubated overnight at 37° C, then 20% Glycerol on 0.5 ml of log culture of bacteria in sterile 1.5 ml test tubes. The two were mixed properly by help of vortex to ensure even distribution. The culture were kept at -80° C for long time storage and further experiments. The process was done according to the protocol described by Sam brook et al., 1989.

3.2.9.2 Stab culture

Sterlized polypropylene vial of 5 ml capacity were filled to two-thirds with molten sterile Luria agar Annexure-A medium. After solidifying, bacterial culture was picked up from Luria broth with the help of inoculation needle and stabbed through the agar. Incubated at 37° C for 18 hours and kept at 4° C for storage. Colonies were confirmed as E. coli & K. pneumonia using biochemical test such as Indole test, Methyl Red test, Voges – Proskauer test and Citrate utilization test. Subsequently, these strains were tested 16Sr RNA and their tolerance to antibiotic resistance and were used for further research exploitation.

            3.2.10 Biochemical characterization of E. coli & K. pneumonia isolates

The IMViC tests are a group of individual tests used in microbiology laboratory testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic or facultative anaerobic rod which produces gas from lactose fermentation within 48 hours. The presence of some coli forms indicate fecal contamination. Except for the lowercase “i”, which is added for ease of pronunciation, each of the letters in “IMViC” stands for one of these tests. “I” is for Indole; “M” is for Methyl red; “V” is for Voges – Proskauer, and “C” is for Citrate (Table 8). These IMViC tests are useful for differentiating bacteria of the family Enterobacteriaceae. E. coli & K. pneumonia isolates were confirmed by IMViC tests, Indole test (to determine the ability of the organism to split indole from the amino acid tryptophan), Methyl-red test (to tests for an evidence of an enteric bacterium). The reaction was developed by Daniel Wilhelm Otto Voges and Bernhard Proskauer, German bacteriologists in 1898 t the Institute for Infectious Diseases, and Citrate utilization test (to check the ability of an organism to use citrate as the sole source of carbon and energy).

3.2.8.1 Indole test

Five drops of Kovac’s reagent Isoamyl alcohol, Paradimethylaminobenzaldehyde, concentrated hydrochloric acid) was added into 2 days old growth of the isolate in 2 ml of Peptone water. A positive result is shown by the presence of a red-pink color in the surface alcohol layer of the broth. A negative result appears Yellow. E. coli is an Indole-Positive bacteria.

3.2.8.2 Methyl- Red (MR) test

Five to six drops of pH indicator Methyl red (MR) reagent was added into a 2 to 5 days growth of the isolate in 3 ml of Glucose-Phosphate Peptone Water. Tube was gently rolled between the palms of the hands to disperse the Methyl red. Enteric that subsequently metabolize pyruvic acid to other acids lower the pH of the medium to 4.2. At this pH, Methyl red turns red. A red color represents a positive test. A yellow color represents a negative a negative test. E.coli is Methyl red-Positive bacteria.

3.2.8.3 Voges-Proskauer (VP) test (Barritt’s Method)

First, 3 ml of 5% solution of ?-naphthol in absolute ethanol, then 1 ml of 40% Potassium Hydroxide (KOH) was added to a five days growth of the isolate in five ml of Glucose-Phosphate Peptone water. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result. E.coli is VP Negative organisms.

3.2.8.4 Citrate Utilization

Slant of Simmons’ citrate agar (Hi Media, Mumbai, India) was inoculated with the presumptive E. coli & K. pneumonia culture and incubated at 37° C for seven days. Growth on the medium without color change is considered as Negative and growth with a development of blue color of the medium is considered as Positive. E. coli is a Citrate Negative organisms.

3.2.11 DNA Extraction from E. coli & K. pneumonia

         To carry out this research we used two different method of isolation genomic DNA for  

           detection of antibiotic resistance genes. First of all, Boiling method used for isolation of    

DNA and after checking the quantity and quality of yielded DNA by running the agarose gel. Run gel for PCR, if we get any specific PCR product band, we go for two other protocols for preserving DNA for long time. Boiling method has some advantages and disadvantages; advantages such as its rapid and less expensive and it gets whole DNA and main disadvantage of Boiling method is that it can’t be kept it for long time (maximum one week). The DNA extraction was set up according to the protocol described by Sam brook et al., 2009.

Boiling method protocol for isolation of DNA

1) 1.5 ml culture is spinned at 8000 rpm for 10 minutes.

2) Discard supernatant in discard bottle (because contain bacteria).

3) Dissolve pellet in 200 µl 1x PBS.

4) Vortex it slightly until pellet dissolves in solution.

5) Centrifuge at 8000 rpm for 10 minutes.

6) Discard supernatant.

7) Dissolve pellet in 200 µl 1x PBS.

8) Centrifuge at 8000 rpm for 10 minutes.

9) Dissolve pellet in 100 µl MilliQ water. 

           10) Boil for 10 minutes, then keep it at room temperature for 10 minutes

           11) Took upper portion for 40 µl.

           12) Store at -20° C (maximum for one week).

In next process, we carry DNA isolation with two different protocol for genomic (Phenol, Chloroform, Isoamyl alcohol) and Plasmid (Plasmid “mini-prep”) to keep DNA for long time for further experiments.

3.2.11.1 Genomic DNA isolation from bacterial cells by PCI method

         The Genomic DNA isolation process described by Sam brook et al., 1989.

1.      Centrifuge the overnight (fresh) culture at 13000 rpm for 10 minutes, and discard     supernatant carefully.

2.      Adding 20µl of cell lysis and vortex it, then add 20µl of proteinase K (50µg/ml) and   

vortex it slightly.

3.      Incubate at 55° C for 90 minutes in water bath (micro tubes should sealed by parafilm).

4.      Add 220µl of PCI solution (saturated Phenol: chloroform: Isoamyl alcohol; 25:24:1)   and then mix it between palms.

5.      Centrifuge at 13000 rpm for 15 minutes and take upper layer to new micro tubes.

6.      Step 4 and 5 is repeated again for a second time.

7.      Add equal amount of Chloroform to upper layer.

8.      Centrifuge at 13000 rpm for 15 minutes and collect aquous phase and transfer to new micro tubes.

9.      Add double volume of Chilled absolute ethanol.

10.  Leave it overnight in -20° C refrigerator (we also can keep it just for 1-2 hours).

11.  Centrifuge it at 13000 rpm for 20 minutes to obtain DNA pellet, discard the supernatant.

12.  Add 300 µl of ice-cold 70% ethanol for washing the pellet.

13.  Centrifuge the microtubes at 13000 rpm for 10 minutes.

14.  Carefully pour off ethanol.

15.  Allow it to dry for 30-45 minutes at 37° C (in incubator).

16.  Add 50µl of hydration buffer (1x MilliQ TE).

17.  Allow it to rehydrate at room temperature for 10 minutes.

18.  Store in 4° C refrigerator.

19.  Next day run on 0.8% agarose gel for checking the quality and quantity of DNA.

3.2.11 Equilibrate of Phenol

·         Phenol used to remove protein from nucleic acid in chromosomal DNA isolation.

·         Cell extraction such as Organic-phenol, CHCl3; high salt; guanidinium HCl.

·         Remove of cell debris such as Proteins, Lipids, Polysaccharides.

·         Concentration of DNA such as Ethanol, Isopropanol; DNA absorbing matrix; CTAB, Sperm dine.

Stepwise procedure to equilibrate phenol.

1.      Heat a water bath to 65° C and place the bottle of phenol in the 65° c water bath to

melt the crystals.

           2.  Add 8-hydroxyquinoline to a final concentration of 0.1% w/v to the phenol. Mix to

                dissolve the 8-hydroxyquinoline.

           3.  Add an equal volume of 1M Tris-HCL (pH 8.0) to the phenol. Mix for 15 minutes.

                Return the bottle to the 65° C water bath. Allow the phases to separate. Siphon off the

                top layer and discard.

           4.  Repeat the procedure as in Step 3 twice.  

           5.  Add an equal volume of 0.5M Tris-HCL (pH 8.0) to the phenol.

                Repeat the procedure as in step 3.

          6.  Repeat the extractions with 0.5M Tris-HCL (pH 8.0) until the aqueous phase is pH 7.8

               (measure with pH meter). Repeat the procedure as in Step 3. Leave approximate 1 cm

              cm layer of 0.5M Tris-HCL (pH 8.0) over the phenol. Add 2-mercaptoethanol to a final

             concentration of 0.2% w/v to the 0.1M Tris HCL (pH 8.0).

         7. The buffer saturated phenol may be stored at 4°C for 1 month for DNA extraction. Test

             the pH periodically and do not use if the pH is

x

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