The Microscope

Protozoa Identification

Regular monitoring of protozoa

A clean, well adjusted microscope is essential for identification of sludge microfauna. When using the microscope remember to: ensure the condenser is setup correctly keep the lenses clean (always use lens paper - ethanol is good for cleaning very dirty lenses) never scratch the lens on the slide, watch from the side when making large adjustments watch from the side when changing from 10X to 40X objectives.

Protozoa cancomprise up to 5% of the

mass of a healthy activatedsludge, with as many as20,000 organisms/ml.

There are over 200 speciesencountered and so

identification to species levelis rarely carried out! They

are divided up into foureasily identifiable groups forthe purpose of diagnosingproblems with an activated

sludge plant.

Changes in protozoal populations can indicate a wide range of operating problemssuch as: toxicity, shock loading, inadequate nutrients and oxygen deficit. Howeverto interpret these it is important to have baseline conditions regarding your ownplant. The figure opposite is typical of an activated sludge plant - construct onefor your own plant as follows:1) Dilute the sample 1:1 with tap water and prepare a wet mount. A Lund cellallows sufficient depth of liquid for protozoal mobility, and can be calibrated toobtain a quantitative assessment of numbers.2) Count five fields of view in a vertical plane.3) Count protozoa in the order: flagellates, free-swimming protozoa, rotifers, stalkedciliates.4) Record each group as numbers/litre mixed liquor and also note the plant sludgeage, loading rate and effluent BOD. Plot them graphically and display the results.5) Carry out the test at least three times each week (it takes only 20 minutes fromreceipt of sample).

Examining Activated Sludge

Filament Identification

Naming filaments

1) On receipt of sample, spread a drop evenly over each of two microscope slides and allow them to air dry, whilst undertaking steps 2 and 3.2) Spread a drop of sample over a third slide, cover with a cover slip and gently press it down.3) Examine under high power and phase contrast, and note: the size and shape of the flocs presence of protozoa and an estimate of the predominant typesThen at a low power note: presence of filamentous bacteria and an estimate of their abundance (on a scale of 0 to 4)4) After examining the wet mount, the air dried slides are used for filament identification. One of them is stained with the Gram stain, theother using the Neisser stain.

Correct identification of the prevalent filament types requires a wetmount which is examined under phase contrast, an air-dried Gram stainand an air dried Neisser stain. A limited number of characteristics areneeded to identify filaments, and most of them can be seen with thewet mount at a magnification of 1,000X. These characteristics are: Branching (present or absent, if present true or false) Filament shape (straight, smooth-curve, bent, chains, coiled, mycelial) Attached growth (present or absent) Sheath (present or absent) Cross walls (present or absent) Shape of cells (square, rectangular, barrel, sausage shaped) Size (length and width of cell in m) Sulphur deposits

In addition to the above, the staining characteristics of the filamentsare determined from the slides which received the Gram stain andthe Neisser stain, which are both examined under direct illumination(bright field).These characteristics are: Gram +ve (organisms stain a blue colour) Gram -ve (organisms stain a red colour; most filaments are gram -ve) Neisser -ve (whole filament stains light brown) Neisser +ve (whole filament stains blue) Neisser +ve granules (filament stains light brown, with dark blue granules clearly visible)

Suctoria RotiferFlagellated Protozoa Nematode

Vorticella Paramecium OperculariaCarchesium

Filament type GramstainNeisser stain Shape & size

(m)Crosswalls SheathFilament Granule

Nocardia

M. parvicella

S. natans

H. hydrosis

021N

0041

+

+

-

-

-

+

-

-

-

-

-

-

+

+

-

-

-, +

-, +

Branched 1.0 x 1.0 - 2.0

Irregular coils 0.6 x 100 -400

Rounded rods 1.4 x 2.0

Straight and rigid 0.5 x 20 - 100

Barrel to ovoid 1.0 - 2.0 x 1.5 - 2.0

Straight, smooth curve or bents1.4 x 1.5 - 2.0

-

-

+

-

+

+

-

-

+

+

-

+

Neisser Stain(+ve purple/blue; -ve yellow/brown)

Nocardia(unstained)

Thiothrix(sulphur granules)

Loading RatesIs your activated sludge plant operating well?If a treatment plant receives adequate organic material forthe biomass present in the reactor (i.e. it has the correct F/Mratio), with enough oxygen, and no toxic substances present,then a healthy sludge will develop.If the F/M ratio is too high, or too low, then the result will besub-optimal effluent treatment. This is illustrated below.

Organic LoadingRate (F/M)

Too Low

Ideal

Too High

Predominant Protozoa

Stalked ciliates and rotifers, with some nematodeworms present.

A large number of protozoa and a wide range ofspecies. Dominated by free-swimming and stalkedciliates.

Large number of flagellates and small,free-swimming ciliates.

Symptoms

Causes

Cures

SSVIThe Test

This chart was prepared by Dr Nigel Horan and the photomicrographs supplied by Dr Louise Hornsby (School of Civil Engineering, University of Leeds).

SSVI120 No SSVI

Attached Growth

Absent Present

Presence of Sheath

Present Absent

Crosswalls Present

Square Rectangular

Barrel

Filament Shape

Straight Smooth Curve

Coiled Mycelial

Euplotes ChilodonellaAspidisca Gram Stain(+ve and -ve)

M. parvicella(gram stained)

O21N(unstained)

S.natans(unstained)

H. hydrosis(unstained)

M. parvicella(unstained)

Activated Sludge Troubleshooting Chart

Yara Industrial, Immingham, N. E. Lincolnshire, DN40 2NSTel: 01469 554711 Email: yarauk.info@yara.com

www.yara.co.uk

Good settling sludge, manyprotozoa in mixed liquor,

especially stalked ciliates, andfree-swimming protozoa with

some rotifers.

Poor settling sludge, manyfilamentous bacteria, dispersedfloc with few stalked protozoabut abundant flagellates and

free-swimming protozoa.Unable to control sludge

blanket in clarifier.

No discernible settlement,highly dispersed floc, few or

no protozoa - mainlyflagellates.

Ideal operating conditions.

Filamentous sludge bulking orfoaming. May be due to: low

dissolved oxygen (DO),nutrient deficiency,

inappropriate F/M, septicity.

Toxic material in influent,organic shock loading,

operating F/M too high, chronicnutrient deficiency.

Continue this operating regime.

Optimise operation ofsecondary sedimentation tank

(use the WRc nomograph)check DO, nutrients and

sulphide levels.

Undertake toxicity evaluation,monitor influent loading,

check nutrient levels.

Flagellatedprotozoa

Small (5-20m)and very fast

moving protozoawith one or more

whip-likeflagella.

Because theymove so fast

they are difficultto see clearly.

Free swimmingciliated protozoa

Much larger(20-100m) andslower swimming

than theflagellates. Withshort, hair-like cilia

which aid theirmobility. Many ofthese protozoa can

be observedcrawling over thesurface of sludge

flocs.

Stalkedciliates

Very easilyrecognisedbell-shapedprotozoa,

attached by astalk to the

sludge floc. Eachstalk can have

one, two ornumerous bells

attached to eachstalk.

RotifersThese are very

large(100-500m),

slow-moving andeasily recognised.

They are notactually protozoa,

but metazoa.

100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -0 -

0 1 2 4 6 8 10 16 18 20 24| | | | | | | | | | |

Flagellatedprotozoa

Free-swimmingciliates

Stalkedciliates

Rotifers

Sludge age (d)Toxicity increasing

Loading Rate increasing

Filaments are identified and named usinga dichotomous key, and those producedby Eikelboom or Jenkins are commonlyemployed. However in the UK a limited

range of filament types are routinelyencountered, and with experience these

are quickly recognised. They are:Nocardia,

Type 021N, Type 0041,

Microthrix parvicella,Sphaerotilus natans and

Haliscomenobacter hydrosis.Their key characteristics are summarised

opposite:

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Yara Industrial offers a rangeof products and dosingcontrollers for biological effluent treatment. The SciSoland Vitamax ranges offer asource of balanced nutrients andtrace metals; Nutriox willprevent the odour and treatabilityproblems caused by hydrogensulphide.

Our customers are supported bya network of field-basedtechnical service staff.

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