Second Annual Progress & Planning Meeting. Quantitative and Qualitative Microbiological Analysis. Neelima Nair and Rachel Lawrence
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1 Second Annual Progress & Planning Meeting Quantitative and Qualitative Microbiological Analysis Neelima Nair and Rachel Lawrence 9 th January 2018
2 INTRODUCTION 52 Probiotic Samples were received from Bangladesh Plated in four different culture media: Agar Bacillus Differential Agar Yeast Malt Agar Agar Colonies were scraped and DNA was extracted Primers were designed for the respective 16s region of the bacteria
3 B. liceniformes (154 bp) B. polymyxa (219 bp) P. putida (170 bp) no band P. denitrificans(118 bp) Nitrosomonas ( no band) Nitrobacter (138 bp) L. lactis (no band bp) L. helviticus (no band 118bp) B. megaterium (111 bp) B. pumilus (227 bp) Eg: Probiotic Sample 28 B.subtilis, B.licheniformis, B. polymyxa, P.putida, P.denitrificans, Nitrosomonas, Nitrobacter, L.lactis, L.helveticus, B.megaterium, B.pumilus, Aspergillus niger, Saccharomycyes cerevisiae
4 B. megaterium (111bp) B. licheniformis (154 bp) Cross-Reaction of 16s Primers 1 7 9
5 Endoglucanase Gene Can Be Used For Identification Of Bacillus subtilis B.sub EN1F CCAGTAGCCAAGAATGGCCAGC 1341bp B.Sub EN1R GGAATAATCGCCGCTTTG 11 L B Ashe, S., Maji, U. J., Sen, R., Mohanty, S., & Maiti, N. K. (2014). Specific oligonucleotide primers for detection of endoglucanase positive Bacillus subtilisby PCR. 3 Biotech, 4(5),
6 Identification Of Saccharomyces cerevisiae Sach F GCGCTTTACATTCAGATCCCGAG 150bp Sach R TAAGTTGGTTGTCAGCAAGATTG B Hana Suranska, Dana Vranova, Jinna Omelkova; 2016; Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains; Brazillian Journal of Microbiology, 47;
7 Bacillus sp. (154 bp) Bacillus sp. (154 bp) Enterococcus faecalis (110 bp) Clostridium butyricum (104 bp) # Bacteria Culture Plates Gel 1. B. mesentericus (Bacillus sp.) Enterococcus Faecalis Clostridium butyricum 2. None 3. Bacillus sp.
8 Bacillus sp. (154 bp) Pediococcus sp. (118 bp) # Bacteria plate Gel 4. None 5. Bacillus subtilis 6. Bacillus sp. Pediococcus sp.
9 B. megaterium (111 bp) Thiothrix (244 bp) Nitrococcus (231 bp) Bacillus liceniformes (154 bp) Lactobacillus acidophilus ( no band) Nitrobacter (138 bp) Sample Bacteria Plate Gel 7. B. subtilis B. liceniformis Nitrosomonas Nitrobacter 8. No Data 9. B. subtilis B. megaterium Thiothrix Nitrococcus
10 L. acidophilus ( no band) L. acidophilus ( no band) Sample Bacteria plate Gel 10. L. acidophilus B. subtilis 11. B. subtilis L. acidophillus
11 L. acidophilus ( no band) Bacillus liceniformes (154 bp) Bacillus megaterium (111 bp) Bacilluspumilus (227 bp) Bacillus amyloliquefaciens (298 bp) Sample Bacteria plate Gel 12. B. subtilis B. liceniformis B. megaterium B. pumilus B. amyloliquefaciens 13. No data 14. L. acidophilus B. subtilis
12 Bacillus sp. (154 bp) L. sporogenesis (227 bp) Bacillus sp. (154 bp) B. liceniformes (154 bp) L. acidophilus ( no band) Nitrobacter (138 bp) Sample Bacteria plate Gel 15. B. mesentericus B. subtilis B. liceniformis L. acidophilus Nitrobacter Nitrosomonas 16. Bacillus sp. L. Sporogenesis
13 B. polymyxa (219 bp) B.liceniformes (154 bp) Nitrosomonas ( no band) Nitrobacter (138 bp) P. denitrificans(118 bp) Bacillus liceniformes (154 bp) Bacillus polymyxa (219 bp) Bacillus megaterium (111 bp) P. putida (170 bp) no band Pseudomonas denitrificans(118 bp) Nitrosomonas ( no band) Nitrobacter (138 bp) Lactobacillus lactis (no band bp) L. helviticus (no band 118bp) Bacteria plate Gel 17. B. subtilis b. liceniformis B. polymyxa B. megaterium B. pumilus P. putida P. denitrificans Nitrosomonas Nitrobacter L. lactis L. Helveticus 18 B. subtilis B. polymyxa B. liceniformis Nitrosomonas Nitrobacter P. Denitrificans
14 B. liceniformes (154 bp) B. megaterium (111 bp) L. acidophilus ( no band) L. plantarum ( 138 bp) Nitrococcus (231 bp) Sample Bacteria plate Gel 19. B. subtilis Nitrococcus 20. Spirulina 21. B. subtilis B. liceniformis B. megaterium L. acidophilus L. plantarum
15 Bacillus sp. (154 bp) Enterococcus faecalis (110 bp) Clostridium butyricum (104 bp) B.subtilis (298 bp) B. liceniformes (154 bp) B.megaterium (111 bp) L. acidophilus ( no band) Sample Bacteria plate Gel 22. B. subtilis B. liceniformis B. megaterium L. acidophilus 23. B. mesentericus (Bacillus sp.) Enterococcus faecalis Clostridium butyricum
16 Bacillus liceniformes(154 bp) Bacillus megaterium (111 bp) L.acidophilus (no band) L.plantarum (138 bp) B.subtilis (298 bp) Sample Bacteria plate Gel 24. B. subtilis 25. B. subtilis B. liceniformis B. megaterium L. acidophilus L. plantarum
17 B. liceniformes (154 bp) B. polymyxa (219 bp) P. putida (170 bp) no band P. denitrificans(118 bp) Nitrosomonas ( no band) Nitrobacter (138 bp) L. lactis (no band bp) L. helviticus (no band 118bp) B. megaterium (111 bp) B. pumilus (227 bp) B.subtilis (298 bp) Nitrobacter (138 bp) Nitrococcus (231 bp) Sample Bacteria plate Gel 26. B. subtilis Nitrobacter Nitrococcus 27. None 28. B. subtilis b. liceniformis B. polymyxa B. megaterium B. pumilus P. putida P. denitrificans Nitrosomonas Nitrobacter L. lactis L. Helveticus
18 B. megaterium (111 bp) Thiothrix (244 bp) Nitrococcus (231 bp) Bacillus sp. (154 bp) B. thermodenitrificans (291 bp) Sample Bacteria plate Gel 29. None 30. Bacillus sp. B. thermodenitrificans 31. B. subtilis B. megaterium Thiothrix Nitrococcus
19 L. acidophilus ( no band) Bacillus sp. (154 bp) B. liceniformes (154 bp) L. acidophilus ( no band) Nitrobacter (138 bp) Nitrosomonas ( no band) Sample Bacteria plate Gel 32. No data 33. B. mesentericus B. subtilis B. licheniformis L. acidophilus Nitrobacter sp. Nitrosomonas sp. 34. L. acidophilus B. subtilis
20 Nitrobacter (138 bp) Nitrococcus (231 bp) L. acidophilus (no band) Sample Bacteria plate Gel 35. Lactobacillus B. subtilis 36. B. subtilis Nitrobacter sp. Nitrococcus
21 B. megaterium (111 bp) Thiothrix (244 bp) Nitrococcus (231 bp) Sample Bacteria plate Gel 37. No data 38. B. subtilis Saccharomyces cerevisiae 39. No data 40. B. subtilis B. megaterium Thiothrix Nitrococcus
22 Sample Bacteria plate Gel 41. Lactobacillus B. subtilis 42. B. subtilis 43. No data
23 B. polymyxa (219 bp) B. liceniformes (154 bp) Nitrosomonas ( no band) Nitrobacter (138 bp) P. denitrificans(118 bp) Nitrobacter (138 bp) Nitrococcus (231 bp) Sample Bacteria plate Gel 44. No data 45. No data 46. B. subtilis Nitrobacter Nitrococcus 47. No data 48. No data 49. B. subtilis B. polymyxa B. liceniformis Nitrosomonas Nitrobacter P. denitrificans
24 B. liceniformes (154 bp) B. polymyxa (219 bp) B. megaterium (111 bp) B. pumilus (227 bp) P. putida (170 bp) no band P. denitrificans(118 bp) Nitrosomonas ( no band) Nitrobacter (138 bp) L. lactis (no band bp) L. helviticus ( 118bp) Sample Bacteria plate Gel 50. B. subtilis B. licheniformis B. polymyxa B. megaterium B. pumilus, P. putida P. denitrificans Nitrosomonas Nitrobacter L. lactis L. helveticus 51. B. subtilis 52. No data
25 Summary We were able to identify B. subtilis and Saccharomyces cerevisiae using species-specific primers. Cross-reaction of primers Either the primers made were not specific or there has been contamination in the sampling procedure or during the production of these PHPs. New species-specific primers to be tested.
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