Method for the Microbiological Examination of Foods

1. 1205 324 Food Industrial Microbiology Method for the Microbiological Examination of Foods

2. Method Rapid Method Direct epifluorescent filter technique (DEFT) Electrical impedance Enzyme-linked immunosorbent assay(ELISA) Traditional method • Plate counts • Membrane filtration • Most probable number • Direct microscopic count • Dye reduction tests • Indicator

3. Plate count method Standard plate count (SPC) Aerobic plate count (APC) Total bacteria count (TBC) Total viablecount (TVC) “Live”

4. Plate count method • Diluent • 0.85%NaCl • 0.1% peptone • Phosphate buffer • Medium • Elective medium • Selective medium • General • Petri dish plate • Replication • Pour plate • Spread plate • Drop plate

5. Diluent Food homogenate Dilution series Medium Plating method Incubate conditions Plate count depends on

6. Selective agent Sodium tellulite Lithium chloride Elective agent Sodium pyruvate Glycine Diagnostic agent Egg yolk Baird-Parker Agar Staphylococcus aureus Opaque zone

7. Plate count method

8. Pour plate

9. Spread plate Number of colony forming units (cfus) ?????

10. Spread plate Number of colony forming units (cfus) ?????

11. ??????? Disadvantage of plate count ???

12. Drop plate • Sample: • Small vol. ≈ 20 μL • Cost

13. Drop plate

14. Check quality of RM & final products • Check condition hygiene • Estimate storage life of products • Determine • Production • Transport • Storage • Determine pathogens Application of plate count

15. Selection of media in food microbiology Adam and Moss (2003)

16. Streak technique http://www.towson.edu/~cberkowe/medmicro/images/streak.gif

17. Filtration 0.45 μm Liquid food • Low number of MO. Large volume of food • Count • Sterilize http://www.biology.lsu.edu/webfac/rgayda/biol1011/Lecture_notesF2004/lecture8.pdf

18. Most probable number (MPN) Multiple tube techniques Most probable number • Pathogen • Number too low • Coliform • Escherichia coli • Staphylococcus aureus • Feacal streptococci

19. Most probable number

20. Direct microscopic count (DMCs) Small sample (0.01 ml) & rapid Optical light microscope Total cell living & dead cells Foods Liquid Semi-solid Microscopic count • Ex. • Milk • Wine • Yogurt starter • Tomato sauce • Howard mold

21. Microscopic count

22. Comparison of sensitivity of method

23. Methylene blue Leuco-methylene blue Dye-reduction test Resazurin (blue) Resorufin (pink) Dihydroresofin (leuco) Triphenyltetrazolium chloride (leuco) Formazan (red)

24. Hygiene indicator • Cross contamination Indicators • Fresh meat • Raw milk • Pasteurized milk

25. ATP : Adenosine triphosphate Synthesis of new cell Active transport (uptake of materials from environment) Movement Light production ATP photometry

26. Luciferin + luciferase + ATP + O2 Oxyluciferin + luciferase + AMP + light ATP photometry Mg2+ 1 ATP light 1 photon

27. Bacteria cell 1 fg of ATP Yeast cell 100 fg of ATP ATP photometry • Limit of ATP photometry102-103 fg ATP/ml fg = femto gram = 10-15 g

28. Break down the non-microbial cells in food Remove non-microbial ATP using ATPase Release ATP from bacteria cell Addition of luciferin & luciferasee Record light emission (ATP photometry) ATP photometry

29. Application Fresh meat Milk Starter culture Test UHT milk Surface contamination ATP photometry

30. Disadvantage Mixed bacteria & yeast cell Dilution Remove cell before ATP measured Filtration Centrifugation ATP photometry

31. Liquid food Filter through membrane Acridine orange : fluorescent dye (fluorochrome) pour through filter Epifluorescent microscopy Count: manual or automatic Direct epifluorescent filter technique (DEFT) • Direct microscopy • Membrane filtration • Vol. of sample • Filter area • Area of microscope field • Number of field

32. Acridine orange bineds to: RNA --- fluorescent orange DNA --- fluorescent green Viable cell RNA > DNA --- orange Non-viable cell DNA > RNA ---green Direct epifluorescent filter technique (DEFT) RNA DNA

33. Direct epifluorescent filter technique (DEFT) Non-viable Viable

34. www.teagasc.ie/.../ 4681/eopr-4681.htm Membrane epiflurescent A rapid technique for the detection of pathogens in food products A rapid technique for the detection of pathogens in food products A rapid technique for the detection of pathogens in food products

35. Membrane epiflurescent

36. Impedance : resistance Bacteria growth ----decrease impedance ----increase conductivity Electrical impedance method Conductance Time DT Detection time 106-107 cells/ml

37. Bactometer Vary temp Small volume Many wells Many samples Automatic Electrical impedance method • Count • Growth

38. Bactometer

39. Antigen – conjugate enzyme Antibody – conjugate enzyme ELISA • Toxin • Staphylocaccal • Botulinum toxin • Mycotoxin • Pathogen • Salmonella • Listeria • S. aureus

40. Antibody Antigen(toxin) Enzyme Alkaline phosphatase (ALP) Horse Radish Peroxidase (HRP) Substrate Tetramethylbenzidine (TMB) + 30% H2O2 Azinobis sulphonic acid (ABTS) o-phenylinediamine (OPD) p-nitrophenyl phosphate Enzyme-linked Immunosorbent Assay Free toxin Labeled toxin Microtitration plate

42. “ELISA”

43. Sandwich-ELISA Salmonella E. coli antibody Antibody-conjugate enzyme Colorless Substrate Color

44. Aflatoxin • flavus • A. nomius • tamarri • A. parasiticus flavus “Aflatoxin” toxin Aspergillus

45. http://msa.ars.usda.gov/la/srrc/aflatoxin/afcrsp.htm

46. Antibody Antigen(toxin) Enzyme Alkaline phosphatase (ALP) Horse Radish Peroxidase (HRP) Substrate Tetramethylbenzidine (TMB) + 30% H2O2 Azinobis sulphonic acid (ABTS) o-phenylinediamine (OPD) p-nitrophenyl phosphate Enzyme-linked Immunosorbent Assay Free toxin Labeled toxin Microtitration plate

47. E E E E Aflatoxin: Colorless Coloration

48. “ELISA” Antibody Aflatoxin (free toxin) Aflatoxin-enzyme labeled (labeled toxin) Substrate E

49. Direct Competitive ELISA Conc. (ppb) 0 5 10 15 20 Aflatoxin E E

50. Direct Competitive ELISA ppb A B C 0 5 10 15 Standard (Aflatoxin) Foods ample (Un-know) A= ?????? ppb B= ?????? ppb C= ?????? ppb