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

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

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

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

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.

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

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

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.

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

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

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.

Filament Shape Smoothly Curved Straight Irregular

Filament Size Filament length Filament width

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

Cell Size Length of cell Width of cell

Cell Septa Septa No septa

Indentations at Septa No indentations Indentations

Sheath Sheath

Sheath Sheath

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

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

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

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

A Simpler Approach Foaming Bulking

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

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

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

Nostocoida limicola

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 -.

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

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

Type 0092 Low F/M and long sludge age

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

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

Sheath

Type 1701

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

Type 0041

Type 0675

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

Thiothrix

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

Sulfur Depositors Type 021N Thiothrix I & II Type 0914 Beggiatoa

Type 021N

Type 021N

Thiothrix

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

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

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

Microthrix

Nocardia

Type 1863

About Nocardia Foaming Very little foaming No foaming

About Nocardia Foaming Less foaming No foaming

Nocardia Count Scribe marks Field of view 5 Intersections

About Nocardia Foaming Foaming

About Nocardia Foaming Foaming Foaming

About Nocardia Foaming Foaming Foaming

About Nocardia Foaming Foaming

About Nocardia Foaming Foaming

About Nocardia

About Nocardia

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.

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?

HINT

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?

Hint

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!