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Identification of Filamentous Bacteria

Identification of Filamentous Bacteria. A Simple Approach. What’s Needed?. A Microscope with phase contrast 10X or 20X Ocular 40X Ocular 100X Ocular (oil immersion lens). Stains. Gram Stain Neisser Stain Sulfur Stain. Gram stain. Separates bacteria into 2 groups Gram (+) and Gram (-)

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Identification of Filamentous Bacteria

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  1. Identification of Filamentous Bacteria A Simple Approach

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

  3. Stains • Gram Stain • Neisser Stain • Sulfur Stain

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

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

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

  7. Slide Preparation • Wet Mount • Smear • dried for staining

  8. Cover glass Drop Slide Wet Mount

  9. Puke Too Hard!

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

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

  12. Straight Irregular Smoothly Curved Filament Shape

  13. Filament length Filament width Filament Size

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

  15. Length of cell Width of cell Cell Size

  16. Cell Septa Septa No septa

  17. Indentations at Septa Noindentations Indentations

  18. Sheath Sheath

  19. Sheath Sheath

  20. True branching Branching

  21. Branching False branching

  22. Attached growth Epiphyte

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

  24. A Simpler Approach • Foaming • Bulking

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

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

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

  28. Nostocoidalimicola

  29. Type 0675Type 0041

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

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

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

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

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

  35. Sheath

  36. Type 1701

  37. Sheath 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) S. Natans Type 1701

  38. Type 0041

  39. Type 0675

  40. Sheath 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) Type 0675 Type 0041 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 H. Hydrossis

  41. Thiothrix

  42. Sheath This one is easy It normally occurs in bundles It’s common cause is also Low F:M Type 1851 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 • Type 021N • Thiothrix I & II • Type 0914 • Beggiatoa

  44. Type021N

  45. Type021N

  46. Thiothrix

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

  48. Sulfur Depositors • 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

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