Nanoparticles are microscopic particles that are between 1 and 100 nanometers in size, and are too small to be seen by the human eye

Nanoparticles are microscopic particles that are between 1 and 100 nanometers in size, and are too small to be seen by the human eye. They can be found in nature, or created by humans, and can have different physical and chemical properties than larger materials.

1. Isolation,Characterization and study of Biotransformation of arsenic resistant bacteria found in water sample                         Shashank                       Sachin Sharma  MSc Microbiology (Final Year) Department of Microbiology M.J.P Rohilkhand University  Bareilly (U.P) 243006 Under the guidance of:- Ayushi Sharma ( faculty of microbiology) M . J . P Rohilkhand University Bareilly

2. AIM/OBJECTIVE Isolation and Characterization and study of Biotransformation of Arsenic Resistant bacteria found in water Sample

3. Table of Content Aim Introduction About Dissertation Procedure/protocol Sample Collection Media Preparation Morphological Study Biochemical Characterization Enzymatic Analysis Molecular Characterization Result Conclusion

4. INTRODUCTION Arsenic is a naturally ocurring element extensively present in the air, water, and land. In its inorganic form, it is very poisonous. Arsenic is highly toxic in its inorganic form. Arsenic has high magnitude of solubility,its removal from contaminated water is very difficult .

5. Arsenic mainly exist in the environment as arsene,elemental arsenic,arsenite and arsenate. Among all these forms only arsenite and arsenate are more abundant in natural environment than the other two. Arsenate and arsenite both are very toxic in nature,arsenite being more toxic than arsenate. to be continued.... .

6. About Dissertation what motivated us to choose this topic….. Arsenic contamination in water is very concerning due to the following reasons:- It can induce various types of cellular damage in biological systems. According to a 2023 survey, 97% of Indian households use some form of water purification, with 44% using reverse osmosis (RO) systems. However, a 2021 survey by the Indian government found that only 8.7% of people use RO purifiers at home. Water is the basic necessity of every individual and they should be provided with clean and safe water just like air. There is a saying that more than 70% of health problems that humans suffer from are due consumption of contaminated food and water.

7. Procedure/protocol Sample collection from different locations. Media preparation(Sodium Arsenite+NAM). Inoculation of water sample and spread it on Agar plates. Incubation at 37*C for 2-3 days. Observation of desired colonies. Pure culturing onto fresh agar plates. Morphological Study(Grams staining) Biochemical Characterization. Enzymatic analysis. Biotransformation testing Molecular testing

8. Sample collection All the water samples were collected in a steriled 50ml falcon tube aseptically. Location from where samples were collected are as follows:-

9. Sample Number Site of Collection Date of Collection Time 1 NAKATIYA RIVER 12-03-2024 11:23 AM 2 PARSAKHERA 15-03-2024 10:45 AM 3 BAKARGANJ LANDFILL 24-03-2024 12:12 PM 4 BOTANICAL GARDEN MJPRU 30-03-2024 12:40 PM 5 MINI BYPASS 05-04-2024 01:19 PM 6 GURUGRAM 10-04-2024 11:57 AM 7 GADDIKHANPUR 14-04-2024 10:37 AM 8 GIRLS HOSTEL MJPRU 22-04-2024 02:57 PM 9 HUGLI RIVER,KOLKATA 29-04-2024 1:49 PM 10 GOMATI RIVER,LUCKNOW 02-05-2024 03:56 PM 11 AGRICULTURE F IE LD 06-05-2024 03:33 PM 12 SHARDA RIVER,TANAKPUR 16-05-2024 02:30 PM

10. 13 IVRI BAREILLY 20-05-2024 09:35 AM 14 PASHUPATI NATH TEMPLE 24-05-2024 12:40 PM 15 RAM GANGA RIVER 30-05-2024 08;16 AM 16 MAHANAGAR 31-05-2024 10:30 AM 17 HARUNAGLA 02-06-2024 03:49 PM 18 ZIMCORBET WATERFALL, RAMNAGAR 07-06-2024 12:39 PM 19 NARIYAVAL 13-06-2024 01:13 PM 20 BAREILLY COLLEGE 18-06-2024 12:27 PM

11. NAKATIYA,BLY PARSKHERA,BLY BAKARGANJ,BLY BOTANICAL GARDEN(MJPRU) MINIBYPASS,BLY GADDHIKHANPUR GIRLS HOSTEL (MJPRU) IVRI, BLY PASHUPATI NATH TEMPLE,BLY RAMGANGA MAHANAGAR FARIDPUR BAREILLY COLLEGE GURUGRAM HUGLI RIVER,KOLKATA SHARDA RIVER,TANAKPUR ZIMCORBET WATERFALL,RAMNAGAR

12. SHARDA RIVER BAKARGANJ DUMPING SITE,BLY HUGLI RIVER GUMTI RIVER NAKATIYA,BLY MAHANAGAR,BLY

13. RAMGANGA RIVER, BAREILLY ZIMCORBET WATYERFALL, RAMNAGAR BOTANICAL GARDEN M.J.P.R.U OVERHEAD WATER TANK HARUNAGLA PASHUPATI NATH TEMPLE BAREILYY

14. Media preparation We prepared 500ppm sodium arsenite nutrient agar media ,where we inoculate our water sample so that we can isolate such bacteria which are resistence to sodium arsenite . The preparation of media are as follows:-

15. 14 grams of nutrient agar 0.25 grams of sodium arsenite mixed it and then autoclaved it to prepared 500ppm sodium arsenite media pouring on sterile petriplates leave it for solidification

16. Inoculation of water sample on prepared plates Inoculation of water sample on prepared plates Spreading of water sample on agar plates water samples Incubate at 37 °C for 2-3 days

17. Observation of colonie and pure culturing of bacteria

18. Pure cultures

19. Morphological study To study the morphology of bacteria ,we performed gram staining .

20. Biochemical Characterization Methyl red(MR) test Voges-proskauer(VP) test Indole test Citrate utilization test Oxidase test Gelatin hydrolysis test T riple sugar iron agar test

21. METHYL RED TEST

22. +VE -VE VP TEST -VE +VE RESULTS INDOLE TEST

23. +VE -VE RESU L TS CITRATE UTILIZATION TEST

24. CATALASE TEST OXIDASE TEST + VE -VE -VE +VE

25. +VE +VE -VE -VE -VE -VE -VE -VE -VE -VE -VE -VE GELATINE HYDROLYSIS TEST

26. Sample numbers Methyl red(MR) test Voges-proskauer(VP) test Indole test Citrate test Oxidase test Gelatin hydrolysis test S-1 +VE -VE -VE -VE +VE -VE S-2 +VE +VE -VE -VE +VE -VE S-3 +VE -VE -VE +VE -VE +VE S-4 +VE +VE -VE +VE +VE -VE S-5 -VE +VE -VE +VE +VE -VE S-6 +VE +VE -VE +VE +VE +VE S-7 +VE -VE -VE -VE -VE -VE S-8 +VE +VE -VE -VE -VE -VE S-9 -VE -VE -VE -VE +VE +VE S-10 -VE +VE -VE +VE +VE -VE S-11 +VE -VE -VE +VE -VE -VE S-12 +VE -VE -VE +VE -VE -VE Biochemicals test table

27. TRIPLE SUGAR IRON AGAR TEST

28. Sample number SLANT BUTT   H2S Lactose/Sucrose Glucose GAS S-1 -VE -VE -VE -VE S-2 -VE -VE -VE -VE S-3 -VE -VE -VE -VE S-4 +VE +VE +VE -VE S-5 +VE -VE -VE +VE S-6 +VE +VE +VE -VE S-7 +VE -VE -VE +VE S-8 -VE -VE -VE -VE S-9 -VE -VE -VE -VE S-10 -VE -VE -VE -VE S-11 -VE -VE -VE -VE S-12 -VE -VE -VE -VE   TRIPLE SUGAR IRON AGAR TEST TABLE

29. The principle of Antibiotic Susceptibility Testing (AST) involves: 1) Isolating bacteria from a sample. 2) Testing antibiotics to see which inhibit bacterial growth. 3) Interpreting results to guide antibiotic treatment decisions . Antibiotic Susceptibility test

30. Samplenumber Doxycycline- Hydrochloride Ciprofloxacin Azitro-mycin Rifam-picin Chloramphe-nicol Cefoxi-tin Meropen-um Ampicilin Penicilin-G Erythromycin Tetracycline S-1 S (22mm zone) S (20mm zone) S (24mm zone) R S (13mm zone) S (24mm zone) S (25mm zone) R S (16mm zone) S (12mm zone) S (17mm zone) S-2 S S S S S S S S R S (12mm zone) S (32mm zone) S-3 R S (16mm zone) S (10mm zone) S (10mm zone) S (20mm zone) R S (10mm zone) R R S (14mm zone) S (15mm zone) S-4 R S (14mm zone) R R S (10mm zone) S (12mm zone) S (23mm zone) S (10mm zone) R S (10mm zone) S (15mm zone) S-5 R S (18mm zone) R R S (10mm zone) R S (10mm zone) R R - - S-6 R S (15 mm zone) R R S (10mm zone) S (10mm zone) S (22mm zone) R R S (10mm zone) S (14mm zone)

31. S-7 R S (13mm zone) S (19mm zone) R S (10mm zone) R S (10mm zone) S (11mm zone) R S (10mm) R S-8 R S (13mm zone) S (12mm zone) S (10mm zone) S (12mm zone) S (17mm zone) S (10mm zone) S (16mm zone) R S (20mm zone) R S-9 S (29mm zone) S (19mm zone) R S (38mm zone) R S (32mm zone) S (37mm zone) S (38mm zone) R R R S-10 R S (14mm zone) S (18mm zone) R S (10mm zone) R S (28mm zone) R R S (17mm zone) S (32mm zone) S-11 R R S (10mm zone) R S (10mm zone) S (12mm zone) S (23mm zone) S (10mm zone) R S (10mm zone) S (14mm zone) S-12 R R R R S (10mm zone) S (23mmzone) S (23mmzone) R R S (10mm zone) S (12mm zone)

32. Enzymatic Analysis Starch hydrolysis test Sample numbers Amylase S-1 -VE S-2 +VE S-3 -VE S-4 -VE S-5 -VE S-6 -VE S-7 -VE S-8 +VE S-9 -VE S-10 -VE S-11 -VE S-12 -VE S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12 (a) (b) (c)

33. Lipid hydrolysis test Sample numbers Amylase S-1 -VE S-2 -VE S-3 + VE S-4 + VE S-5 + VE S-6 + VE S-7 + VE S-8 -VE S-9 + VE S-10 + VE S-11 + VE S-12 + VE S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12 (a) (b) (c)

34. Biotransformation Heavy metals cannot be degraded, but they can be transformed from one oxidation state to another inorganic complex. According to their effect on the environment and human health, bioremediation is one of the most feasible methods for the remediation of soil that is contaminated with organic and inorganic compounds. This process utilizes microorganisms or plants to decontaminate or remove organic and inorganic xenobiotics from the environment. Bioremediation is an environmentally friendly technology solution to overcome heavy metal contamination. The microbe reaction to metal is a remarkably complex process, which is controlled by several factors, such as type of metal, nature of medium, and microbial species, when active uptake of heavy metals (bioaccumulation) and/or passive uptake (adsorption) occurs Microbial cell walls, which mainly consist of polysaccharides, lipids, and proteins, offer many functional groups, including carboxylate, hydroxyl, amino, and phosphate, which can bind heavy metal ions.

35. Silver nitrate test A qualitative silver nitrate (AgNO3) screening technique was used to detect the oxidation of arsenite to arsenate or the reduction of arsenate to arsenite. In nutrient agar plates were supplemented with sodium arsenite and sodium arsenate. A single line streak of the isolated organism across the centre of the agar plate was made and incubated at 37 °C for 48 hrs. After incubation, the plates were flooded with 0.1 M silver nitrate solution. A brownish precipitate indicated the presence of arsenate i n the medium ( arsenite oxidizing bacteria ) and the presence of arsenite were detected by a bright yellow precipitate (arsenate reducing bacteria). 1.69 gram AgNO3 + 100ml distilled water = 0.1M AgNO3 solution

36. Before After Plates were flooded with 0.1M AgNO3 solution Before After 1) 2) 3) 4) 5) 6)

37. Before After After Before 7) 8) 9) 10) 11) 12)

38. Molecular Characterization Microbial Identification using 16S rRNA based molecular method . Sample: S-8 The Sample was found to Paenibacillus cisolokensis strain UICC B-42 16S ribosomal RNA, partial sequence

39. >1021_037_005_PCR_SAMPLE3_16SF_C02.ab1 TCCCCACATAAGCTAGGTGTCAGGGGGTTTCGATACCCTTGGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGA CTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGAC ATCCCGCTGACCGGTCTAGAGATAGGCCTTTCCTTCGGGACAGCGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGCGCAACCCTTGATTTTAGTTGCCAGCACGTAAAGGTGGGCACTCTAGAATGACTGCCGGTGACAAACCGGAGG AAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAGGAGCGAT TCGGAGCGAATCCTTAAAAGCCGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCA TGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGTCGGTGAGGTAACCCGCAAGG GAGCCAGCCGCCGAAGGTGGGGTAGATGATTGGGGTGAAGTCTACAAAGGGTAACCGAAGAGTTTGATCCTGGCTCAGTAAGTCGTAACAA GGTAACCGTGGAGATTAAACCTGGCTCAGTAACCTCGGGCCACCCCACCCGTACACATTAATCCCGGCGCCCGGGGGGTTACCCCCGCTCCCG GCGCAGAGAAAACATCGTTAGATTTCCCCGGTGCCCCACGGAGGTGCCCCCCCTGTTATTCAACACAGATTTTTACGACGCCCCCGCGGGGGG

40. ATATTTATGAACTAATTTTAGTTACAAAAAACGCCCCCCACATATAAATATGGCGTGGGGTGCGGGGGAAAGATGGTCAGAGGTGTTTTTCTTT TCTAAGGACCACACAAAAAAAAAATTTTCTCTCTGTGTTGTCTCTCTTCCGTGGAAAAAAATATTATCAAAAACCGCCCGCCCTACCCCCCCCCG GTGGGGTTTTCAGGCAACAAATTTACGGGAGAGGGGAGAAAAA >1021_037_006_PCR_SAMPLE3_16SR_C02.ab1 ACCGTCGGTCTCCCAGGGCGGGAATGCTTAATGTGTTAACTTTCGGCACCAAGGGTATCGAAACCCCTGACACCTAGCATTCATCGTTTACGGC GTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGCCCAGAAAGTCGCCTTCGCCACTGGTGTT CCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTTTCCTCTTCTGCACTCAAGCCTTGCAGTTTCCCGTGCTCCCCGGGGTTGAG CCCCGGGTTTAGACACAAGACTTACAAAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTCGCCCCCTACGTATTACCGCGGC TGCTGGCACGTAGTTAGCCGGGGCTTTCTTCTCAGGTACCGTCACCCAAAGAGCAGTTACTCTCCTTGGCGTTCTTCCCTGGCAACAGAGCTTT ACGACCCGAAGGCCTTCCTCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCT GGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATG

41. CGCCGCAGGCCCATCTGCAAGTGACAGCTTGCGCCGCCTTTCCCGGCCCCCTCATGCGAGAAGGCCGCCTATCCGGTATTAGCTCACGTTTCCG TCAGTTATCCCGGTCTTGCAGGCAGGTTGCCTACGTGTTACTCACCCGTCCGCCGCTAAGCTTCAGAGAAGCAAGCTTCTCCTCAGCTCCGCTC GACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCAGATAACAAAACTTAAAAGGTTACCTGGTTACGACTTACTGGGCCAGGATC AAACTCTACGGGTAACTTGTTACGAATTTATGGGTCCGGATAAAACTCTACAGATAACTTTTTACAATTGATAAGCCAAAAAAAAATATACTCTT TCTTGTTTTACTTCTGGGAGAAGAGAGAGAAAAAACCGCGACCTCTCCACCTTGTGTGTGTTGGAGCAACATACGGGGGGAGGCGAAAAAAA TTCCGGGGGCGACCCCGA

42. Sample: S -9 T he Sample was found to Paenibacillus cisolokensis strain UICC B-42 16S ribosomal RNA, partial sequence . >1021_037_007_PCR_SAMPLE4_16SF_D02.ab1 GCCCCAAAAAACACCCATTCGCCCAAGAGACGATTGGAGGCTGGGTCCTTGAGACGTGGCTTCCGGAGCTAACGCGTTAAATCG ACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCTGGAATCCTGTAGAGATACGGGAGTGCCTTCGGGAATCAGAACA CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCC AGCACGTAATGGTGGGAACTCAAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCC TTACGGCCAGGGCTACACACGTGCTACAATGGCGCGTACAGAGGGCTGCAAGCTAGCGATAGTGAGCGAATCCCAAAAAGCGC GTCGTAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGCAAATCAGAATGTTGCGGTGAATA CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCG TTTACCACGGTGTGATTCATGACTGGGGTGAAGTCGTACAAAGGGGAAACCGGAAGAGTTTGATCCTGGCTCAGGAAGTCGTAA CAAGGTAACCGGACAGTTTGATCCTGGCTCACCAAGGCGTAACCACGGAAACCGTACAGATTGATCCGGTCTCCCGGGATCTTA ACCCCGTACCCAGAAATTTTGATCCGGACTCTTTAAAGCGAATACCCGGGGTCAGAGCCGGGGATTCCTCTTCTATTTATACCCAA CCCCCGGAGCGGGGTTTAACGCACCGGATTTCCCATTACGCGTCGACACCCCCCATATAACCGGGCGTGTGGGGACAAAAATCA CGCAAGGTTCTTCTTGGGAGATAGAGCAAAAGAAGAGGTGATATCACCCACACTTTCCCCCTCGGTGAAGGGGTGTAAACACAC GACGCGTCTTCTCAGCCCCGGTGGGGGTGTTGACGGAGGCCCCCAGGGGGGAAAATCCCCCCGGGGGGCCCCCAGAGAAGGG GAGGGGGGGGGGGTTCCTTGGGGGGGGTATGGTCTCCCAACAT

43. >1021_037_008_PCR_SAMPLE4_16SR_D02.ab1 TCCGTGGCGAATTCCCAGGGCGGGTCGATTTAACGCGGTTAGCTCCGGGAAGCCACGGTCTCAAGGACACAGCCTCCAAATCGA CATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGG GCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCT GGACAGTTTTAAATGCAATTCCCAGGTTGAGCCCGGGGCTTTCACATCTAACTTATCCAACCGCCTGCGTGCGCTTTACGCCCAGT AATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGAGTAACGTCACA GCTGGCAGTTATTAGCTACCAACCTTTCCTCCTCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTG CATCAGGGTTTCCCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGAT CATCCTCTCAGACCAGCTAGGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCCACCTGGGTTCATCCAATCGC GCAAGGCCCGAAGGTCCCCTGCTTTCCCCCGTAGGGCGTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCGACTGGG CAGATCCCCAGGCATTACTCACCCGTCCGCCGCTCGCCGGCAAAAGTAGCAAGCTACTTTCCCGCTGCCGCTCGACTTGCATGTG TTAGGCCTGCCGCCAGCGTTCAATCTGAGCCAGATTCAAACTCTACGGTTACCTTGTTACGACTTCCTGAGCCAGGATCAAACTCT ACGGTTACCTTGTTACGACTTACTGAGCCAGGATCAAACTCTACGGGTACCTGGTACAACTACTGAGCCAGGAACAGAACTTACG GTTACCTGTTACGACTGCGCGGGCAGAGAGGATGTCTACG

44. gDNA and 16S Amplicon QC data: Fig 1: gDNA loaded on 0.7 % Agarose gel Lane Description: L: DNA Marker; Sample 3 is S-8 Sample 4 is S-9 S-8 S-9

45. P CR Amplification conditions: 16S rRNA based molecular method DNA: 1 μl 16S Forward Primer: 400ng 16S Reverse Primer: 400ng dNTPs (2.5mM each): 4 μl 10X Taq DNA polymerase Assay Buffer: 10 μl Taq DNA Polymerase Enzyme (3U/ μl): 1 μl Water: X μl Total reaction volume: 100 μl

46. Result s The bacteria identified after performing various test were:- E.coli Staphylococcus aureus Aeromonas veronii Pseudomonas fluoresecens E nterobacter cloacae E ntrobacter aerogenes E nterococcus faecalis Paenibacillus cisolokensis

47. Conclusion Thus form this entire study, it can be concluded that all the isolated samples that is S1-S12,which were identified as Bacillus sp .,Cocci sp .,Pseudomonas sp ., Enterobacter Sp. can tolerate arsenite concentration of 500ppm.These arsenic resistant bacteria can be used as a novel pathway for the bioremediation of arsenic.

48. Future Aspects

49. Acknowledgement I want to sincerely thank everyone who has assisted me in successfully completing this project. I want to start by expressing my gratitude to Miss Ayushi Sharma , my mentor, for her unwavering support, insightful advice, and motivation during the process. Additionally, I would like to thank Dr. Pankaj Kumar Arora and Prof. Alok Shrivastava , Head of Department, Department of Microbiology, for providing all the facilities needed for this project, as well as Dr.Sanjay Patel and Dr.Charanjeet Kaur , who both worked in the Department of Microbiology, for their contributions to my success. I appreciate Mr. Mushtak Ali's help. SACHIN SHARMA SHASHANK