1. Identification of Filamentous Bacteria
A Simple Approach
Introduction:
Who I am.
How I got started.
Why the simple approach is important.

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

3. Stains Gram Stain Neisser Stain Sulfur Stain
Staining procedures are provided in the packet.

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
No explanation needed

5. Neisser stain Separates bacteria in 2 groups Neisser (+) - blue
Neisser (+) and Neisser (-)
Distinguishes those filaments with cells that contain granules that accumulate polyphosphate
Neisser (+) - blue
Neisser (-) - brownish
No explanation needed.

6. Sulfur stain Separates bacteria into 2 groups sulfur deposits
no sulfur deposits
No explanation needed.

7. Slide Preparation Wet Mount Smear dried for staining
Collect the sample from the same place all the time.
Always use the same dilution.

8. Wet Mount Cover glass Drop Slide
If the drop is too big the cover slip will float and you will have too much depth.
You should dab the edges of the cover slip with paper towel to absorb some of the fluid.

9. Too Hard! Puke This can also occur under toxic conditions.
This slide is courtesy of Michael Geraldi.

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. Typical Observation Filament Shape Filament Size Cell shape Cell size
Cell Septa
Indentations
Sheath (present/absent)
Branching (true/false)
Epiphyte (attached growth)
Motility
No explanation needed.

12. Filament Shape
Smoothly
Curved
Straight
Irregular

13. Filament Size
Filament length
Filament width

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

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
Sheath

20. Branching True branching
With true branching the branches are continuous (like a tree). In other words, fluid could flow freely throughout all the branches.

21. Branching False branching
With false branching there is not continuation between branches. It’s like the branches were just glued there.

22. Epiphyte Attached growth
Epiphyte (attached growth) is bacteria attached to the sheath perpendicular to the filament wall.

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

24. A Simpler Approach
Foaming
Bulking

25. BULKING FILAMENTS Major 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. Nostocoida limicola

29. Type 0675 Type 0041
These two are Gram variable. This means some of the cells stain + and some stain -. This usually due to the difference in the age of the cells.
OO41 and 0675 look just alike except one is larger.
Generally the portion of the filament that is within the floc stains Gram +. The portion outside the floc stains Gram -.

30. BULKING FILAMENTS Neisser Stain Neisser (+) Neisser (-) (bluish)
(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 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 Type 1851 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. Type 021N

45. Type 021N

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

50. Microthrix

51. Nocardia

52. Type 1863

53. About Nocardia
Foaming
Very little foaming
No foaming

54. About Nocardia
Foaming
Less foaming
No foaming

55. Nocardia Count
Scribe marks
Field of view
5 Intersections

56. About Nocardia
Foaming
Foaming

57. About Nocardia
Foaming
Foaming
Foaming

58. About Nocardia
Foaming
Foaming
Foaming

59. About Nocardia
Foaming
Foaming

60. About Nocardia
Foaming
Foaming

61. About Nocardia

62. About Nocardia

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

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

65. HINT

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

67. Hint

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