1. Identification of Filamentous Bacteria A Simple Approach

2. What’s Needed? n A Microscope with phase contrast –10X or 20X Ocular –40X Ocular –100X Ocular (oil immersion lens)

3. Stains n Gram Stain n Neisser Stain n Sulfur Stain

4. Gram stain n Separates bacteria into 2 groups –Gram (+) and Gram (-) n Determined by cell wall structure n Gram (+) - purple –Bacterium cell wall retain purple color n Gram (-) - pink –Bacterium cell wall lose purple color

5. Neisser stain n Separates bacteria in 2 groups –Neisser (+) and Neisser (-) n Distinguishes those filaments with cells that contain granules that accumulate polyphosphate n Neisser (+) - blue n Neisser (-) - brownish

6. Sulfur stain n Separates bacteria into 2 groups –sulfur deposits –no sulfur deposits

7. Slide Preparation n Wet Mount n Smear –dried for staining

8. Wet Mount Slide Drop Cover glass

9. Too Hard! Puke

10. Filamentous Bacteria n Normal Permanent residents of activated sludge n Not dominant under normal conditions n Beneficial n Single cell units under normal conditions n Forms filament under certain conditions n Problematic when numerically dominant

11. Typical Observation n Filament Shape n Filament Size n Cell shape n Cell size n Cell Septa n Indentations n Sheath (present/absent) n Branching (true/false) n Epiphyte (attached growth) n Motility

12. Filament Shape Straight Smoothly Curved Irregular

13. Filament Size Filament length Filament width

14. Cell Shape Cocci (round) (oval) Bacillus (rod-shaped) (square) (rectangle) (discoid)

15. Cell Size Length of cell Width of cell

16. Cell Septa Septa No septa

17. Indentations at Septa No indentations Indentations

18. Sheath Sheath

19. Sheath

20. Branching True branching

21. Branching False branching

22. Epiphyte Attached growth

23. Motility n The ability to swim n There is only one filament that is motile n Beggiatoa

24. A Simpler Approach n Foaming n Bulking

25. BULKING FILAMENTS Major Characteristics n Staining characteristics –Gram (Neisser, PHB) n Sheath (with or w/o epiphyte) n Sulfur deposits n Motility n Cell shape

26. BULKING FILAMENTS Gram Stain Gram (+) (purple) Gram (-) (pink)GramVariable Nostocoida limicola Type 0041 Type 0675 Type 1851 All the rest

27. Nostocoida limicola n The only Gram (+) filament that causes bulking only n Does not cause foaming n Purple “beaded necklace”

28. Nostocoida limicola

29. Type 0675 Type 0041

30. BULKING FILAMENTS Neisser Stain Neisser (+) (bluish) Neisser (-) (brownish) Nostocoida limicola Type 0092 All others

31. Nostocoida Neisser (+) (low F/M, presence of organic wastes)

32. Type 0092 Low F/M and long sludge age

33. Characteristics n Sheath (with or w/o attached growth) n Sulfur Depositors n Motility n Branching (true or false)

34. Sheath n type 1701 n type 0041 n type 0675 n type 1851 n Sphaerotilus Natans n Thiothrix I & II n Haliscomenobacter hydrossis

35. Sheath

36. Type 1701

37. Sheath S. Natans Type 1701 They look alike Both have sausage-shaped cells S. Natans is larger S. Natans exhibits false branching Type 1701 often has attached growth (if you can’t figure the difference, it doesn’t matter because the cause for both is the same - Low DO, also simple carbohydrates and organic acids)

38. Type 0041

39. Type 0675

40. Sheath Type 0041 They both look alike Type 0041 is larger Both usually has attached growth (Don’t bother to measure, they have a similar cause - Low F:M, long MCRT, Nitrogen and Phosphorus deficiency) H. Hydrossis The sheath is difficult to detect The filaments are very thin Resembles pins in a pin cushion Associated with low DO, low F:M and nutrient deficiency

41. Thiothrix

42. Sheath Type 1851 This one is easy It normally occurs in bundles It’s common cause is also Low F:M Thiothrix I & II This one is also easy Sulfur granules are usually present The common cause is septic wastes, wastes with an appreciable amount of sulfides and/or organic acids, and wastes deficient in nitrogen

43. Sulfur Depositors n Type 021N n Thiothrix I & II n Type 0914 n Beggiatoa

44. Type 021N

46. Thiothrix

47. Sulfur Depositors Type 021N Cells are shaped like stacked hockey pucks Indentations at the cell septa Thiothrix Cells are rectangular shaped with no indentations at the cell septa Beggiatoa This is easy. This filament is the only filament that “swims”. Type 0914 This filament has rectangular sulfur granules, the others have spherical granules

48. Sulfur Depositors n The common cause of these filaments are: –the presence of reduced sulfur compounds (septic wastes) –wastes deficient in nitrogen –the presence of organic acids

49. FOAMING FILAMENTS Gram Stain Gram (+) (purple) Gram (-) (pink) Microthrix parvicella Nocardia Type 1863

50. Microthrix

51. Nocardia

53. About Nocardia No foaming Very little foaming Foaming

54. About Nocardia Foaming Less foaming No foaming

55. Nocardia Count 5 Intersections Scribe marks Field of view

56. About Nocardia FoamingFoaming

57. Foaming Foaming Foaming

58. Foaming Foaming Foaming

59. Foaming Foaming

60. Foaming Foaming

63. Most Important! n Keep a process chart of treatment system parameters. n Measure the parameters consistently and routinely. n Measure the parameters when the system is running properly.

64. THIS IS A TEST! n The treatment system is experiencing foaming. n You determine that there are filaments present in the foam. n The filament is Gram (+) n It sort of looks like purple spaghetti. WHAT IS IT?

65. HINT

66. THIS IS ANOTHER TEST! n The treatment plant is experiencing foaming. n You determine that there are filaments in the foam* n The filament is Gram (-), Neisser (-), and contains no sulfur. WHAT IS IT?

67. Hint

68. YET ANOTHER TEST! n The treatment plant is having problems with bulking sludge. n After examining a wet mount of mixed liquor, the operator observes excessive amounts of filamentous bacteria. n The filaments are Gram (-), Neisser (-), show a (+) test for sulfur, has no sheath or attached growth. n The strange thing is, the sulfur granules are rectangular shaped!