3.1 used for constant temperature incubation and shaking of

3.1 Materials

 

3.1.1 Chemicals, and molecular biology chemicals

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All the chemicals used were of analytical grade. They were purchased either from Merck, New England Biolab, Hi Media. The nutrients medias used like Luria broth, Luria agar and all the Antibiotics were bought from Hi Media Laboratories, Mumbai India. All the media and reagent which used and prepared for culturing and identification of E. coli & K. pneumonia and other step of research mentioned in Appendix 1. Agarose (Low EEO) was bought from Genie company.

Fine chemicals such as NAOH, Tris buffer, Glucose, Tryptone, Hcl, Tris base, EDTA, Potassium acetate, Glacial acetic acid Isopropyl alcohol etc. was purchased from Qualigens, Merck (India). Chemicals like SDS, Mgcl2 and Pyruvic acid was bought from Sigma Aldrich Company, St Louis, USA. Absolute Ethanol, DTT, Bromophenol blue, Glycerol, Glycine, Acrylamide, bis-acrylamide, Ammonium persulphate, TEMED was bought from Merck, Mumbai, India. All Taq DNA polymerase, dNTPs, and ladders were purchase from New England biolabs. For the purification of  PCR product was purchased from Hi media. Gene specific primers were synthesized from different companies, India.

3.1.2 Instrumentation

1.      Autoclave (model NSW 227), was used for sterilizing all lab ware that could withstand high temperature. Reagents (including ultrapure water) and bacterial growth media was also autoclaved sterlized prior to use.

2.      Bench Top Centrifuge (Eppendrof- model 5415D), 13000 rpm maximum force with 24 tubes of 1.5 ml.

3.      Clinical Incubator (Universal), used for constant temperature incubation of cultures on plates.

4.      Cold Rack (Vest frost), used for short term storage of culture for constant temperature incubation and shaking of cultures on minimal media broth.es and reagents at 4° C.

5.      Deep Freezer (-20° C) Blue star, and (-80° C) Ultra low Refrigerator Sanyo for long term storage of enzymes and DNA.

6.      Environmental Shaker (Sanyo), used for constant temperature incubation and shaking of cultures on minimal media broth.

7.      Electronic Balance (Precisa), for weighing purpose.

8.      Gel Electrophoresis Horizontal (Banglore Genei), used for agarose gel electrophoresis.

9.      PCR (Bio-rad) for the amplification of target genes.

10.  Pipette Aid, equipped with 0.2 mm filter ( Eppendrof), employed for sterile routine pipetting of culture media and solutions.

11.  Micropipettes, adjustable (Eppendrof), are used to measure small volumes.

12.  Spectrophotometer, employed for routine assessment of culture growth through the measurement of culture’s turbidity or optical density at 400-600nm.

13.  Vortex Mixer (Bangalore Genie), used for proper mixing the contents of tube.

3.1.3 Chemical and Biological Decontamination and Aseptic Techniques

All labwares used in this study was thoroughly chemically decontaminated prior to use. The cleaning procedure varied according to the chemical resistance of each material. In general the decontamination process, included final storage into double sealed polyethylene bags. Glass and plastic lab ware was decontaminated using similar washing procedure, adjusted for their chemical resistance. In general, they were treated with 3M  Hcl, thoroughly rinsing with ultra pure water, dried and stored in double plastic bags until used.

All lab wares and solutions used in the experiments were also biologically decontaminated. Most of the materials and solutions were sterlized by autoclaving. The standard autoclave program for liquids involves heating to 121° C for 20-25 minutes at 1atm (psi). Some of the material like powder free gloves, plastic plates, etc. that cannot be sterlized by autoclaving was purchased sterile (radio logically sterlized).

All handling and processing of samples of this research was conducted in clean air environment refereed as clean lab. Any critical sample manipulations were done by using contamination free equipments, lab wares and reagents to minimize contamination.  

3.2 Methods

            3.2.1 Collection of water samples and isolation of presumptive E. coli .   

                     pneumoniae strains

      Water samples were collected from 22 km Delhi stretch of the Yamuna River, starting from upstream of the Wazirabad Barrage to downstream of the Okhla Barrage, apart from Sewage water and other water bodies, during a period of 7 Months ( June 2016- December 2016) from various points along the river Yamuna traversing through the National Capital Territory of Delhi. These sampling site were Wazirabad, ISBT (Kashmiri Gate), Sarai Kale Khan, Okhla Barrage, DND Flyway, Rajghat, Faridabad waste water, ITO bridge, Hindon river, Rohini canal, Rohini sewage water, Punjabi Bagh sewage water, Hasanpur village sewage water, Rohini ground water & Sarai Kale Khan ground water. One hundred eighty  samples were collected from the different-2 sites of the Seven sites of Yamuna river, Three sites of sewage water, Two sites of Ground water, One sites of Canal water & Two sites of other water bodies, at depth of 1.5 feet & Ground water from 100 feet depth. Sampling collection was performed in sterile 5ml screw-capped tubes. Isothermal box were used for collection of samples. Prior to sample collection, Isothermal box were washed with dilute acid followed by distilled water and were dried. Before taking water samples, the Isothermal box were rinsed three times with the water to be collected. Analysis of water samples was conducted after collection to avoid unpredictable changes in water sample. Samples were brought in Isothermal box. As the samples have to be processed within 12 hour of collection, which were transported to the laboratory on ice.

     3.2.1.1 A brief account of reason & anthropological activities associated with each

                 sampling site are given below.

These sites have grouped into five groups and details of all the groups are given below:

 

Group-1                                                                                  

            3.2.1.2 Wazirabad barrage

Because of spiritual faith, the bathing in River Yamuna of Wazirabad barrage is very common. Bathing, especially mass bathing, significantly contributes disease-causing pathogens in the river water and enhance the bacterial load. The religious activities e.g. offering flowers, milk, sweets etc. into the river water further increase organic load in the river. The other activities associated with bathing are clothes washing. This activity contributes inorganic, organic and biological contaminants into the river water beside detergents. Excessive presence of detergent caused significant foaming at the site of turbulence. Foaming not only hampers the oxygen diffusion rate in the river water, essential for self-purification, but also affect various biological activities including food chain.

3.2.1.3 ISBT (Kashmiri Gate) 

Due to the non-existence of sanitary facilities in rural areas and urban areas, especially in slum clusters, a large section of the population use either catchment area or directly to the river for open defecation. The activity contributes organic pollution and pathogens in the river water. People in these areas generally have the practice of open defecation and discharge of sewage in the Yamuna river catchment area due to this the water quality of the river is continuously deteriorating. Water contaminated with fecal matter causes diarrhea & pneumonia and cholera are a very common disease of the slum areas in this area.

3.2.1.4 Sarai kale khan

 

Farmers are using large quantities of chemical fertilizer, insecticides, pesticides, to increase short-term crop yields or keeping the soil productive, without knowing the exact quantities are required. It is estimated that about one-half of every metric ton of fertilizer or pesticides applied to fields never even makes it into plant tissue, but instead, ends up evaporating or being washed into local waterways. The excess amount of fertilizer use entered the soil, ground and surface water bodies and pollutes them and during the rainy season by runoff, it pollutes the lakes, ponds, and rivers and causes eutrophication, which decreases the dissolved oxygen level and threatens animal and plant health.

3.2.1.5 ITO Bridge

At ITO this is the central point of Yamuna in Delhi. Most of the drainages being combined before this point. Disposal of sewage effluents is big problems almost in every big city. It cannot be simply disposed of due to their microbiological and chemical characteristics. A Million liters of untreated sewage is disposed of in the Yamuna. The organic matters and microorganisms are the main constituents of the domestic waste. Besides these, total salts, chlorides, nutrients, detergents, oil & grease etc. are also contributed by the domestic sources. There are numerous unauthorized colonies exist in various urban centers. Due to non-availability of sewerage system in these colonies, the night soil is collected, transported and dumped either in drains, tributaries or directly into the river without any treatment. During last few years because of the proliferation of Jhuggi Jhonpri settlement, this activity increased significantly and now become a major non-point source of river water pollution. 

3.2.1.6 Rajghat

Because of close proximity of Yamuna Temple at Rajghat, In people (Hindus) bathe in rivers due to religious convictions and beliefs at Rajghat and dump holy materials and related materials along with domestic solid waste in rivers. River Yamuna is among one of the holiest rivers in India and people frequently take a mass bath in the river that  severely affects the water quality. Deterioration of river water quality may injure the health of the people taking the dip and also the population downstream which use the river as a source of water for drinking, bathing and other domestic purposes.

3.2.1.7 DND Flyway

 

DND Flyway site of River Yamuna receives the significantly high amount of organic matter, which generally, originates from domestic sources. This organic waste requires oxygen, depletion of dissolved oxygen in river water. The oxygen depletion for its biodegradation & while doing so leads do not only affects the biotic community of the river but also affects its self-purification capacity. In DND Flyway site, the load of organic matter is so high that it consumes the entire dissolved oxygen available in river water.  Organic, inorganic and toxic pollutants generated from agricultural and industrial sources are accumulated near the source during dry seasons and get mixed with river water posing threat to aquatic life during monsoon or percolated to ground water and making water unfit for human consumption.

3.2.1.8 Okhla Barrage 

Cremation in Yamuna River and on its banks is also one of the reasons of river water pollution. Every day cremations are performed, most on wood pyres that do not completely consume the large numbers of the body. Along with the remains of these traditional funerals, there are hundred more who cannot afford cremation and whose bodies are simply thrown into the Yamuna. The absence of adequate cremation facilities is contributing to a large number of partially unburnt corpses floating down the Yamuna.

Group -2

3.2.2 Rohini canal

 

The cattle at most of the towns & villages near the Rohini canal are regularly taken toward the river for drinking and bathing. It is estimated that total cattle population in the Rohini canal uses for bathing and watering purposes directly. These cattle activities impart substantial impact on water quality. This occurs not only through direct discharge of urine, dung and washed off organic-inorganic materials but the bottom sediments are also churned up because of cattle wading.

 Group-3

 

 3.2.3 Hindon River

 

After independence, rapid industrialization occurred in the Yamuna river basin. There are large clusters of industries established at Sonepat, Delhi, Baghpat, Ghaziabad, & other places. The categories of industries discharging wastewater into Hindon river includes Pulp & Paper, Sugar, Distilleries, Textiles, Leather, Chemical, Pharmaceuticals, Oil Refineries, Thermal Power Plants, food etc.

Group-4

 

3.2.4 Faridabad Waste Water

 

The categories of industries discharging wastewater into Yamuna river includes Pulp & Paper, Sugar, Distilleries, Textiles, Leather, Chemical, Pharmaceuticals, Oil Refineries, Thermal Power Plants, food etc.

Group-5

 

3.2.5 Rohini Sewage, Punjabi Bagh (sewage), Hasanpur village (sewage)

 

Untreated and open drainages have produced conducive breeding for mosquitoes, flies, rodents, insects and other diseases.

Untreated sewage has led to stinking and source of foul smell.

Group-6

 

3.2.6 Rohini(Ground water)

 

Malfunctioning septic systems have resulted in contamination of ground water.

         3.2.6.1 Sarai kale Khan (Ground water)           

Toxic food farming leading to various diseases like as vomiting, gastroenteritis, diarrhea & Pneumonia.

           3.2.7 Preparation of Media:

           Different bacterial media were prepared for culturing bacterial strains.

Luria broth, Luria agar, MacConkey agar, EMB agar powder and Glycerol was         purchased from Hi-Media (India).

3.2.7.1 Luria-Bertani medium (LB Medium)

20 grams of Luria-Bertani medium (Appendix 1 ) was added in 900ml of double distilled water and dissolved: the volume was made up to 1 liter with double distilled water. Media was sterilized by autoclaving at 15lb/sq.inch (at 121°c) in the autoclave for 20 minutes.

3.2.7.2 Luria Agar Medium (LA Medium)

35 grams of Luria Agar medium (Appendix 1) was added in 900 ml of double distilled water and dissolved: the volume was made up to 1 liter with double distilled water. Media was sterilized by autoclaving at 15lb/sq.inch (at 121°c) in the autoclave for 20 minutes.

3.2.7.3 MacConkey Agar Medium (MCA Medium):

51 grams of MacConkey Agar medium was added in 900 ml of double distilled water and dissolved: the volume was made up to 1 liter with double distilled water. Media was sterilized by autoclaving at 15lb/sq.inch (at 121°c) in the autoclave for 20 minutes.

3.2.7.4 Eosin Methyl Blue medium (EMB Medium)

For 1 liter of medium, 36 grams of Eosin Methyl Blue medium was added in 900 ml of

double distilled water. Media was sterilized by autoclaving at 15 lb/sq.inch (at 121°c) in the autoclave for 20 minutes.

 

3.2.8 Pure Cultures

3.2.8.1 Screening of E. coli & K. pneumoniae strains

Water samples were subcultures on MacConkey Agar (culture medium designed to grow gram negative bacteria and differentiate them for lactose fermentation) Hi Media, Mumbai, India, to select lactose fermenting colonies. Pink colonies in MacConkey plates were considered as lactose fermenting colonies and selected for transfer to Eosin Methylene Blue agar (EMB) also known as ” Levine’s formulation (Hi Media, Mumbai, India). This medium is important in medical laboratories for distinguishing pathogenic microbes in a short period of time. Eosin Methylene Blue (EMB) is selective for gram-negative bacteria. It is a blend of two strains, Eosin and Methylene blue in the ratio of 6:1. A common application of this stain is in the preparation of EMB agar, a differential microbiological medium, which inhibits the growth of gram-positive bacteria and provides a color indication distinguishing between organisms that ferment lactose (e.g., E. coli & K. pneumonia) and those that don’t (Salmonella Shigella).Organism that ferment lactose display “nucleated colonies” colonies with dark centers. Lactose fermentation produces acids, which lower the pH. This encourages dye absorption by the colonies, which are now colored purple black. Lactose non-fermenters may increase the pH by deamination of proteins. This ensures that the dye is not absorbed. On EMB, if E. coli is grown, it will give a distinctive metallic green sheen (due to the meta chromatic properties of the dyes, E. coli movement using flagella, and strong acid end-products of fermentation). Some species of Citrobacter and Enterobacter will also react this way to EMB.