Transcript 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 (-)
Determined by cell wall structure
 Gram (+) - purple

– Bacterium cell wall retain purple color

Gram (-) - pink
– Bacterium cell wall lose purple color
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

Sulfur stain

Separates bacteria into 2 groups
– sulfur deposits
– no sulfur deposits
Slide Preparation
Wet Mount
 Smear

– dried for staining
Wet Mount
Cover glass
Drop
Slide
Too Hard!
Puke
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
Filament Shape
Straight
Smoothly
Curved
Irregular
Filament Size
Filament length
Filament width
Cell Shape
(oval)
(discoid)
(square)
(round)
(rod-shaped)
(rectangle)
Cocci
Bacillus
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
Branching
False branching
Epiphyte
Attached growth
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)
Nostocoida limicola
Gram
Variable
Gram (-)
(pink)
Type 0041
Type 0675
Type 1851
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
BULKING FILAMENTS
Neisser Stain
Neisser (+)
(bluish)
Neisser (-)
(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
Sheath
w/epiphyte
w/o epiphyte
Type 1701
Type 0041
Type 0675
Type 1851
S. Natans
H. Hydrossis
Thiothrix I & II
Type 1701
Type 0041
Type 0675
Type 1851
S. Natans
Thiothrix I & II
H. Hydrossis
Type 1701
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)
Type 0041
Type 0675
Sheath
Type 0675
Type 0041
H. Hydrossis
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)
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
Thiothrix
Sheath
Type 1851
This one is easy
It normally occurs in bundles
It’s common cause is also Low F:M
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
Thiothrix
I & II
Sulfur Depositors
Type 021N
 Thiothrix I & II
 Type 0914
 Beggiatoa

Type
021N
Type
021N
Thiothrix
Sulfur Depositors
Type 021N
Thiothrix
Beggiatoa
Type 0914
Cells are shaped like stacked hockey pucks
Indentations at the cell septa
Cells are rectangular shaped with
no indentations at the cell septa
This is easy. This filament is
the only filament that “swims”.
This filament has rectangular sulfur
granules, the others have spherical granules
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
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
Count
25.0
20.0
15.0
Foaming
Foaming
10.0
5.0
4/28
4/25
4/22
4/19
4/16
4/13
4/10
4/7
4/4
4/1
0.0
About Nocardia
Count
25
Foaming
20
15
Foaming
Foaming
10
5
5/31
5/28
5/25
5/22
5/19
5/16
5/13
5/10
5/7
5/4
5/1
0
About Nocardia
Count
50
45
40
35
30
25
20
15
10
5
0
Foaming
Foaming
6/28
6/25
6/22
6/19
6/16
6/13
6/10
6/7
6/4
6/1
Foaming
About Nocardia
Count
7/31
7/28
7/25
7/16
7/13
7/10
7/7
7/4
7/1
7/22
Foaming
Foaming
7/19
100
90
80
70
60
50
40
30
20
10
0
About Nocardia
Count
200
Foaming
Foaming
150
100
50
8/31
8/28
8/25
8/22
8/19
8/16
8/13
8/10
8/7
8/4
8/1
0
About Nocardia
Count
9/28
9/25
9/22
9/19
9/16
9/13
9/10
9/7
9/4
9/1
100
90
80
70
60
50
40
30
20
10
0
10/31
10/28
10/25
10/22
10/19
10/16
10/13
10/10
10/7
10/4
10/1
About Nocardia
Count
100
90
80
70
60
50
40
30
20
10
0
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!