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Thewater is the most basic and necessary element in life. In North India, the Yamuna River is the main tributary of the Ganga River. It is approximately 1370 kilometers long. The Yamuna River is named after the Yamunotri Glacier in Uttar Pradesh. Tons and Giri are key tributaries of the Yamuna River and the primary supply of water in hilly areas. Before joining with the Ganges at Allahabad, the Yamuna runs through the states of Delhi, Haryana, and Uttar Pradesh. On its banks are world-famous towns such as Delhi, Mathura, and Agra. Because of the huge volume of wastewater discharge, one of the most polluted rivers in India is the Yamuna, particularly in the Delhi area.

Membrane filtration test

Membrane filtration is a water sample testing procedure. Water is drawn through a specific porous membrane intended to catch microorganisms bigger than 0.45 m in this method.

Samples of Delhi and Okhla Yamuna river were taken and poured into the fennel which had a porous membrane which filter outs the water sample.

Nutrient broth was poured and was kept in shaker incubator for 15 minutes over the membrane filter so to collect any kind of bacteria presence over membrane. It was incubated for 48 hours at 37 C. Colonies were observed in Delhi sample which were further tested in Endo agar. Endo agar is a selective media that stimulates gram-negative bacterial growth and inhibits gram-positive growth. It also contains lactose for fermentation and a dye to show pH changes.

The water sample drawn out of fennel was tested by directly measuring 0.1ml of sample into nutrient agar for growth of any kind of colonies that might have passed through the membrane. The sample obtained from Delhi Yamuna showed growth of microbiological colonies after incubating it for 24 hours while Ohkla sample showed no such growth even after incubation period of 48 hours. Delhi sample was furthermore tested for presence of coliform bacteria.

Figure 1:  Membrane filtration techniques being performed with the help of Membrane Filter Unit.

Figure 2:  Poring of samples in Membrane filter unit.

Figure 3:  Incubator for 15 minutes.

The tests showed positive result for presence of coliform bacterial In Delhi sample

Most probable number

The most probable number (MPN) methodology is a method for determining the quantity of bacteria in a food or water sample. In this procedure, replicating sections of the original sample are cultivated to evaluate the presence or absence of microorganisms in each part.

Coliform Identification

The Most Probable Number (MPN) test was carried out by inoculating 5 test tubes (with inverted Durham tubes) each containing 10 ml of diluted sample to 10 ml of double strength MacConkey Broth, 1 ml diluted sample to 10 ml of single strength MacConkey Broth, and 0.1 ml diluted sample to 10 ml single strength MacConkey Broth. The test tubes were incubated for 24 to 48 hours at 37 degrees Celsius. The test tubes were then examined for turbidity, acidity, and gas production.0.1ml of sample from MacConkey Broth tubes was removed, and the tubes were inoculated in 10ml of BGBB (Brilliant Green Bile Broth), and turbidity, acid, and gas generation in the tubes were examined. In this test, materials from tubes with positive findings for gas and acid generation were streaked on EMB (Eosin Methylene Blue agar). The petri plates were incubated, and the development of fecal coliform was detected.

Pseudomonas and Aeromonas species identification

1 mL of sample water was inoculated in 9 mL of peptone water containing 1% NaCl (w/v) and pH 8.6 adjusted with sodium hydroxide. After incubating at 37C for 24-48 hours, the cultures were streaked on Glutamate Starch Phenol Red agar (GSP) and incubated for another 24-28 hours.

Salmonella and Shigella species identification

1 mL of sample water is inoculated in 9 mL of 1 percent NaCl (w/v) water. A total of 5 test tubes were serially diluted. The produced dilution was then streaked on Hektoen Enteric Agar and incubated at 37C for 24-48 hours.

E. coli identification

4 mL of tryptophan broth was placed in sterilized test tubes. Results are awaited.

Figure 4: Results obtained in Most Probable Number

Serial dilution

The act of taking a sample and diluting it via a succession of standard quantities of sterile diluent, which can be either distilled water or 0.9 percent saline, is known as serial dilution. A tiny, measured volume of each dilution is then used to create a series of pour or spread plates.

Multiple test tubes are taken, here, 21 for 3 samples.

It is done repeatedly by micropipette, water sample is taken in a test tube in labelled test tube A, B, C, D, E, F, G for 3 samples. A being 10 ml of pure sample and G being the 10-6.

Serial dilutions were done on S1, S2 and S3 samples respectively after which they were kept fin incubator for 24 hours at 37 C. The plates contained nutrient agar which is a nutrient medium that is used to cultivate microorganisms and support the development of a broad variety of non-fastidious organisms. Nutrient agar is popular because it can support the development of a wide range of bacteria and fungi and includes numerous nutrients required for bacterial growth.

After the incubation periods Petri plates were observed and growth was observed in all plated samples, Delhi sample observing the highest growth of microbes amongst all samples collected. The colonies were isolated in another round of petri plates to get pure culture of colonies. They were then tested for identification of microorganisms obtained.

 

Figure 5:Illustration of Serial dilution.

Morphological test

Gram staining is essential for phenotypic characterization of microorganisms. The staining process distinguishes Bacteria species based on cell wall structure. Gram-positive cells contain a thick peptidoglycan coating and appear blue to purple when stained. Gram-negative cells are distinguished by a thin peptidoglycan coating that stains red to pink.

The Gram stain, bacteriology's most extensively used staining process, is a sophisticated and differential staining procedure. The cell wall composition of organisms in the Domain Bacteria is distinguished using a series of staining and decolorization methods. Gram-positive bacteria have extensive coatings of peptidoglycan in their cellwalls (90 percent of the cell wall). These are purple-colored. Gram-negative bacteria have thin peptidoglycan coatings (10 percent of the wall) and a high lipid content. These are pink-colored. Because Archeae and Eukaryotes lack peptidoglycan, this staining method is not employed. Applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant(Gram's Iodine), quick decolorization with alcohol, acetone, or a combination of alcohol and acetone, and finally counterstaining with safranin are the four fundamental processes of the Gram Stain.

Figure 6: Growth of various colonies obtained in Nutrient Agar media.

Biochemical tests

Biochemical tests are those that are done on various bacteria to identify them based on their biochemical activity toward various biochemical chemicals.Microbial biochemistry tests minimize the time necessary to identify bacteria, save expenses, and assure or improve the accuracy of an unknown sample's identification. It is the newest trend in microbial identification.

Properties	S1(Agra)	S2(Delhi)	S3(Ohkla)
Shape	rod	rod	rod
Motile (Motile/Nonmotile)	Non- motile	motile	motile
Spore (Non sporing/sporing)	sporing	sporing	sporing
Gram staining	-ve	-ve	+ve
Catalase	+ve	+ve	+ve
Oxidase	-ve	+ve	-ve

   Table 1: Results of various biochemical tests performed on isolated stains of microbes.
   
Figure 7: Gram staining results of various microbes under microscope. 

Collection of samples

Samples were taken from different areas of Yamuna River in the areas of Delhi, Ohkla and Agra city.

They were labelled S1 for Agra sample, S2 for Delhi sample and S3 for Ohkla river.

Samples were collected in month of February and march in a reusable sterilized water container.

Physio-chemical test

AGRA (Sample – 1)

The hydrogen ion concentration is measured using the pH scale, which quantifies the acidity and alkalinity of water. The pH of water samples varied from 7.3 to 7.7 of sample. The pH of water sample S1 is somewhat alkaline.

DELHI (Sample – 2)

The pH of water sample was between 7.7 to 7.9. It is highly alkaline for a drinking water.

OHKLA (Sample – 3)

The pH of water sample was 7.2 detect on pH meter which is alkaline.

Thetests results indicated presences of microbial bodies in water sample obtained from all three locations. MPN test confirmed presences of coliform bacteria in sample of Delhi Water tests via MFT. Identification of microbes is still under process viz various biochemical tests.

The MFT test were not considered to be true as the units present in lab were of expiry dates hence true results could not be obtained and the broth sample spilled during the two trials, but the filtered water was collected and tested properly.

1. Anurag Kumar, Jane Claryn Benjamin, Arti Kumari and Hemant Kumar, Isolation and Identification of Bacterial Strains from Yamuna River at Allahabad District in Uttar Pradesh, India, Department of Industrial Microbiology, Jacob Institute of Biotechnology and BioEngineering, 2 Faculty of Health Sciences, 3College of Forestry, SHUATS, Allahabad - 211007, U.P., India. 2. Nupur Raghav, Pooja Saraswat, Janendra Nath Srivastava, Rajiv Ranjan, Metagenomics Study of Bacterial Communities from Yamuna River water of city of Taj, Agra, Cold Spring HarborLaboratary, March 2022. 3. AJS (2015) An overview on drinking water supply in Agra, Agra Jal Sanasthan March 2015. 4. APHA (1998) Standard Methods for the Examination of Water and waste water, 19th Eds. American Public Health Association, Washington DC. 5. BIS (Bureau of Indian Standards) (2012) “IS: 10500:2012”, Drinking Water – Specification (Second Revision), Drinking Water Sectional Committee, FAD25, India, 1-11. 6. Pooja Upadhyay, Arushi Saxena, PammiGauba, Biological analysis of Yamuna River, Department of Biotechnology, Jaypee Institute of Information Technology, Noida A-10, Sector-62, Noida, Uttar Pradesh-201307.