Identification of Identification of Filamentous BacteriaFilamentous Bacteria

A Simple ApproachA Simple Approach

What’s Needed?What’s Needed?

A Microscope with phase contrastA Microscope with phase contrast– 10X or 20X Ocular10X or 20X Ocular– 40X Ocular40X Ocular– 100X Ocular (oil immersion lens)100X Ocular (oil immersion lens)

StainsStains

Gram StainGram Stain Neisser StainNeisser Stain Sulfur StainSulfur Stain

Gram stainGram stain

Separates bacteria into 2 groupsSeparates bacteria into 2 groups– Gram (+) and Gram (-)Gram (+) and Gram (-)

Determined by cell wall structureDetermined by cell wall structure Gram (+) - purpleGram (+) - purple

– Bacterium cell wall retain purple colorBacterium cell wall retain purple color Gram (-) - pinkGram (-) - pink

– Bacterium cell wall lose purple colorBacterium cell wall lose purple color

Neisser stainNeisser stain

Separates bacteria in 2 groupsSeparates bacteria in 2 groups– Neisser (+) and Neisser (-)Neisser (+) and Neisser (-)

Distinguishes those filaments with cells Distinguishes those filaments with cells that contain granules that accumulate that contain granules that accumulate polyphosphatepolyphosphate

Neisser (+) - blueNeisser (+) - blue Neisser (-) - brownishNeisser (-) - brownish

Sulfur stainSulfur stain

Separates bacteria into 2 groupsSeparates bacteria into 2 groups– sulfur depositssulfur deposits– no sulfur depositsno sulfur deposits

Slide PreparationSlide Preparation

Wet MountWet Mount SmearSmear

– dried for staining dried for staining

Wet MountWet Mount

Slide

Drop

Cover glass

Too Hard!Too Hard!

Puke

Filamentous BacteriaFilamentous Bacteria

Normal Permanent residents of Normal Permanent residents of activated sludgeactivated sludge

Not dominant under normal conditionsNot dominant under normal conditions BeneficialBeneficial Single cell units under normal Single cell units under normal

conditionsconditions Forms filament under certain conditionsForms filament under certain conditions Problematic when numerically dominantProblematic when numerically dominant

Typical ObservationTypical Observation

Filament ShapeFilament Shape Filament SizeFilament Size Cell shapeCell shape Cell sizeCell size Cell SeptaCell Septa Indentations Indentations

Sheath Sheath (present/absent)(present/absent)

Branching Branching (true/false)(true/false)

Epiphyte Epiphyte (attached growth)(attached growth)

MotilityMotility

Filament ShapeFilament Shape

Straight SmoothlyCurved

Irregular

Filament SizeFilament Size

Filament length

Filament width

Cell ShapeCell Shape

Cocci

(round)

(oval)

Bacillus

(rod-shaped)(square)

(rectangle)

(discoid)

Cell SizeCell Size

Length of cell

Width of cell

Cell SeptaCell Septa

Septa

No septa

Indentations at SeptaIndentations at Septa

No indentations

Indentations

SheathSheathSheath

SheathSheath

Sheath

BranchingBranching True branching

BranchingBranching

False branching

EpiphyteEpiphyte

Attached growth

MotilityMotility

The ability to swimThe ability to swim There is only one filament that is There is only one filament that is

motilemotile BeggiatoaBeggiatoa

A Simpler ApproachA Simpler Approach

FoamingFoaming BulkingBulking

BULKING FILAMENTSBULKING FILAMENTSMajor CharacteristicsMajor Characteristics

Staining characteristicsStaining characteristics– Gram (Neisser, PHB)Gram (Neisser, PHB)

Sheath (with or w/o epiphyte)Sheath (with or w/o epiphyte) Sulfur depositsSulfur deposits MotilityMotility Cell shapeCell shape

BULKING FILAMENTSBULKING FILAMENTS

Gram StainGram Stain

Gram (+)Gram (+)(purple)(purple)

Gram (-)Gram (-)(pink)(pink)

GramGramVariableVariable

Nostocoida limicolaNostocoida limicolaType 0041Type 0041

Type 0675Type 0675

Type 1851Type 1851

All the restAll the rest

Nostocoida limicolaNostocoida limicola

The only Gram (+) filament that The only Gram (+) filament that causes bulking onlycauses bulking only

Does not cause foamingDoes not cause foaming Purple “beaded necklace”Purple “beaded necklace”

NostocoidaNostocoidalimicolalimicola

Type 0675Type 0675Type 0041Type 0041

BULKING FILAMENTSBULKING FILAMENTS

Neisser StainNeisser Stain

Neisser (+)Neisser (+)

(bluish)(bluish)

Neisser (-)Neisser (-)

(brownish)(brownish)

Nostocoida Nostocoida limicolalimicola

Type 0092Type 0092All othersAll others

NostocoidaNostocoida

Neisser (+)Neisser (+)

(low F/M, presence(low F/M, presence

of organic of organic

wastes)wastes)

Type 0092Type 0092Low F/M Low F/M

and longand long

sludge agesludge age

CharacteristicsCharacteristics

Sheath (with or w/o attached Sheath (with or w/o attached growth)growth)

Sulfur DepositorsSulfur Depositors MotilityMotility Branching (true or false)Branching (true or false)

SheathSheath

type 1701type 1701 type 0041type 0041 type 0675type 0675 type 1851type 1851 Sphaerotilus NatansSphaerotilus Natans Thiothrix I & IIThiothrix I & II Haliscomenobacter hydrossisHaliscomenobacter hydrossis

SheathSheathSheath w/epiphyte w/o epiphyte

Type 1701Type 0041Type 0675Type 1851S. NatansH. HydrossisThiothrix I & II

Type 1701Type 0041Type 0675Type 1851

S. NatansThiothrix I & IIH. Hydrossis

Type 1701Type 1701

SheathSheath

S. NatansType 1701

They look alikeBoth have sausage-shaped cells

S. Natans is largerS. 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 bothis the same - Low DO, also simple

carbohydratesand organic acids)

Type 0041Type 0041

Type 0675Type 0675

SheathSheath

Type 0675Type 0041

They both look alikeType 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. HydrossisThe sheath is difficult to detectThe sheath is difficult to detect

The filaments are very thinThe filaments are very thinResembles pins in a pin cushionResembles pins in a pin cushion

Associated with low DO, low F:M andAssociated with low DO, low F:M andnutrient deficiencynutrient deficiency

ThiothrixThiothrix

SheathSheath

Type 1851 This one is easyIt normally occurs in bundles

It’s common cause is also Low F:M

ThiothrixI & II

This one is also easySulfur granules are usually presentThe common cause is septic wastes,

wastes with an appreciable amount of sulfides and/or organic acids, and wastes deficient in nitrogen

Sulfur DepositorsSulfur Depositors

Type 021NType 021N Thiothrix I & IIThiothrix I & II Type 0914Type 0914 BeggiatoaBeggiatoa

TypeType021N021N

TypeType021N021N

ThiothrixThiothrix

Sulfur DepositorsSulfur Depositors

Type 021NType 021N

Cells are shaped like stacked hockey pucksCells are shaped like stacked hockey pucksIndentations at the cell septaIndentations at the cell septa

ThiothrixThiothrix

Cells are rectangular shaped withCells are rectangular shaped withno indentations at the cell septano indentations at the cell septa

BeggiatoaBeggiatoaThis is easy. This filament isThis is easy. This filament is

the only filament that “swims”.the only filament that “swims”.

Type 0914Type 0914 This filament has rectangular sulfurThis filament has rectangular sulfurgranules, the others have spherical granulesgranules, the others have spherical granules

Sulfur DepositorsSulfur Depositors

The common cause of these The common cause of these filaments are:filaments are:– the presence of reduced sulfur the presence of reduced sulfur

compounds (septic wastes)compounds (septic wastes)– wastes deficient in nitrogenwastes deficient in nitrogen– the presence of organic acidsthe presence of organic acids

FOAMING FILAMENTSFOAMING FILAMENTS

Gram StainGram Stain

Gram (+)Gram (+)(purple)(purple)

Gram (-)Gram (-)(pink)(pink)

Microthrix parvicellaMicrothrix parvicella

NocardiaNocardia

Type 1863Type 1863

MicrothrixMicrothrix

NocardiaNocardia

Type 1863Type 1863

About NocardiaAbout Nocardia

No foamingNo foaming

Very little foamingVery little foaming

FoamingFoaming

About NocardiaAbout Nocardia

FoamingFoaming

Less foamingLess foaming

No foamingNo foaming

Nocardia CountNocardia Count

5 Intersections5 Intersections

Scribe marksScribe marks

Field of viewField of view

About NocardiaAbout Nocardia

0.0

5.0

10.0

15.0

20.0

25.0

4/1

4/4

4/7

4/10

4/13

4/16

4/19

4/22

4/25

4/28

Count

FoamingFoaming FoamingFoaming

About NocardiaAbout Nocardia

0

5

10

15

20

25

5/1

5/4

5/7

5/10

5/13

5/16

5/19

5/22

5/25

5/28

5/31

Count

FoamingFoamingFoamingFoaming

FoamingFoaming

About NocardiaAbout Nocardia

05

101520253035404550

6/1

6/4

6/7

6/10

6/13

6/16

6/19

6/22

6/25

6/28

Count

FoamingFoaming

FoamingFoamingFoamingFoaming

About NocardiaAbout Nocardia

0102030405060708090

100

7/1

7/4

7/7

7/10

7/13

7/16

7/19

7/22

7/25

7/28

7/31

Count

FoamingFoaming FoamingFoaming

About NocardiaAbout Nocardia

0

50

100

150

200

8/1

8/4

8/7

8/10

8/13

8/16

8/19

8/22

8/25

8/28

8/31

Count

FoamingFoaming FoamingFoaming

About NocardiaAbout Nocardia

0102030405060708090

100

9/1

9/4

9/7

9/10

9/13

9/16

9/19

9/22

9/25

9/28

Count

About NocardiaAbout Nocardia

0102030405060708090

100

10/1

10/4

10/7

10/1

0

10/1

3

10/1

6

10/1

9

10/2

2

10/2

5

10/2

8

10/3

1

Count

Most Important!Most Important!

Keep a process chart of treatment Keep a process chart of treatment system parameters.system parameters.

Measure the parameters Measure the parameters consistently and routinely.consistently and routinely.

Measure the parameters when the Measure the parameters when the system is running properly.system is running properly.

THIS IS A TEST!THIS IS A TEST!

The treatment system is The treatment system is experiencing foaming.experiencing foaming.

You determine that there are You determine that there are filaments present in the foam.filaments present in the foam.

The filament is Gram (+)The filament is Gram (+) It sort of looks like purple It sort of looks like purple

spaghetti.spaghetti.WHAT IS IT?WHAT IS IT?

HINTHINT

THIS IS ANOTHER TEST!THIS IS ANOTHER TEST!

The treatment plant is The treatment plant is experiencing foaming.experiencing foaming.

You determine that there are You determine that there are filaments in the foam*filaments in the foam*

The filament is Gram (-), Neisser The filament is Gram (-), Neisser (-), and contains no sulfur.(-), and contains no sulfur.

WHAT IS IT?WHAT IS IT?

HintHint

YET ANOTHER TEST!YET ANOTHER TEST! The treatment plant is having problems with The treatment plant is having problems with

bulking sludge.bulking sludge. After examining a wet mount of mixed After examining a wet mount of mixed

liquor, the operator observes excessive liquor, the operator observes excessive amounts of filamentous bacteria.amounts of filamentous bacteria.

The filaments are Gram (-), Neisser (-), show The filaments are Gram (-), Neisser (-), show a (+) test for sulfur, has no sheath or a (+) test for sulfur, has no sheath or attached growth.attached growth.

The strange thing is, the sulfur granules are The strange thing is, the sulfur granules are rectangular shaped!rectangular shaped!