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| Isolation and Identification of Microbes From Various Areas of River Yamuna | |||||||||||||||||||||||||||||
| Paper Id :
16791 Submission Date :
2022-12-15 Acceptance Date :
2022-12-22 Publication Date :
2022-12-25
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| Abstract |
Several tests were done over a period to determine any presences of pathogen in water bodies of Delhi, Okhla and Agra. The chosen river was Yamuna due to its increased level of toxin and pollutionamongstall the other river present in India. The sample was gathered and tests in lab by multiple methods to detect any pathogen that may cause disease if the water is consumed. MPN tests showed positive result, MFT showed majority of pathogens are larger than .45 μm. Serial dilution showed multiple growth of colonies in all samples.
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| Keywords | Yamuna River, E.coli, Water Pollution, Salmonella spp, Isolation, Identification. | ||||||||||||||||||||||||||||
| Introduction |
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.
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| Objective of study | To detect the quality of Holy River Yamuna used in daily life of humans and animals. |
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| Review of Literature | According to the Centre for Science and Environment, raw sewage, industrial runoff, and rubbish dumped into the river account for 75 to 80 percent of the river's pollution, which totals more than 3 billion liters of waste every day (Mishra, 2010 and Martínez et al., 2009). According to a United Nations research, freshwater is progressively becoming an issue, with about 900 million people suffering every year from diarrhoea and an equivalent amount suffering from sickness caused by different worms. Unclean water is the most serious global population issue. The samples were taken from the surface of different areas from the Yamuna river– Agra(27.176911,78.044095), Delhi(28.624509,77.252785) and Okhla (28.571960,77.293848). The samples were gathered between march and April several times from sites S2 and S3 as MFT were performed multiple times. Each time the samples were gathered the odour, colour varied from last time. Serial dilution, Membrane filtration were done several time, each time showing positive growth of varies colonies.
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| Main Text |
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 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.
   Figure 7: Gram staining results of various microbes under microscope.
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| Sampling |
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.
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| Result and Discussion |
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.
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| Conclusion |
The tests done above confirmed that the water sample is unfit for drinking purpose not only for people, but also for animals. Nearby areas around the river tend to depend on this very water for purpose of drinking, washing, bathing, etc, which needs to be decourged as soon as possible. The toxin present in water is hard to clean and can cause multiple diseases in human if the water is consumed by mistake. The test confirmed the presences of coliform meaning presences of fecal in the water. The industrial waste is also a prime reason for alarming level of water pollution in the Yamuna River. If, such water is used for irrigation it may introduce toxin in crops and might infect the entire crop resulting in unfit for consuming. With the alarming level of presences of pathogen is detection in every 0.1 ml of sample the Yamuna water is quite contaminatedand not suitable for any other such purpose. |
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| References | 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. |
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