Microscopic Examination of

Activated Sludge

Educational Objectives
Upon completion of this course, the operator should be able to use the microscope to view microorganisms
present in activated sludge, understand their differences, metabolism and correct distribution to achieve
optimum wastewater treatment. In addition, the operator will understand the importance of the microorganism
groups as they relate to determining the food to microorganism (f/m) ratio and settling characteristic. Once
the operator becomes proficient with these operational tests, the wastewater treatment plant effluent will be of
higher quality.

I. Abstract. Amoeboid - Amoeboid protozoa has extremely flexible mem-

This operator education course explains differences between
basic types of microorganisms present in activated sludge,
their metabolism and correct distribution to achieve optimum
wastewater treatment. In addition, the Settling test and food to
microorganism (f/m) ratio is explained and sample calculations
are provided to aid the operator in optimizing treatment of the
wastewater prior to discharge. Optimizing the treatment will as-
sist the operator to be in compliance with its National Pollutant
Discharge Elimination System (NPDES) Permit. Questions and
answer section is also provided to determine if the operator un-
derstood the material.
branes that allow food to be absorbed through them. They move
throughout the MLSS by the movement of protoplasm within the
cell. Generally, Amoeboids are found in high numbers in “young”
MLSS and is associated with poor settling sludge.
Flagellates - Flagellate protozoa move throughout the MLSS by
the corkscrew movement of the tail (Flagella) which extends from
their round or elliptical cell configuration. Generally, Flagellates
are found in high numbers where the Amoeboid population is low,
and a high organic load (BOD). As the MLSS ages and exhibits
good settling, the Flagellate population is reduced to one-eight
(1/8) of the protozoans.

II. Keywords.
Mixed Liquor Suspended Solids (MLSS) - The volume of activated
sludge in the aerator.
Activated Sludge - A brownish floc-like substance made up largely
of organic matter derived from the sewage. This substance is pop-
ulated by millions of bacteria and other forms of biological life.
Solids Settling Test - A test to determine the settling capability of
activated sludge after 30 minutes.
Ciliates - Ciliate protozoa move throughout the MLSS by rotat-
ing hair-like membrane (cilia) which cover all or part of their cell
membrane. Additionally, they use the cilia to pull food into their
gullet. There are two types of Ciliates, free swimming and stalked
Ciliates. Ciliates are associated with fair to good settling of the
activated sludge.
Settleometer - A clear cylinder with calibration markings of 50 mL
intervals, starting at the bottom, and ending at the mouth with
1000 mL.

Food to Microorganism (F/M) ratio - A ratio of food per day to
the amount of microorganisms calculated from the aeration tank.
Heterotrophic and Autotrophic bacteria - Bacteria which are re-
sponsible for purifying the wastewater.
Protozoa - Microorganisms that metabolize different nutrients
and clarifying the wastewater by consuming the bacteria.
III. Introduction
Wastewater is a mixture of solids and water, with water represent-
ing 99.9% of wastewater. That means that only .1% represents a
pollution and health problem, unfortunately that is more than
sufficient to cause human health problems. Wastewater must be
sufficiently treated to remove possible threats to human health.
The typical wastewater treatment plant three portions, Pre-
liminary treatment, biological, secondary treatment and tertiary
treatment.

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 The preliminary treatment of the raw wastewater,
termed, influent, includes physical or chemical removal of
grit and screenings to be disposed of in a landfill. Grit and
screening represents the inorganic portion of the wastewa-
ter. The removal of the inorganic portion, allows efficient
breakdown of the organic pollutants, such as, Carbona-
ceous Biochemical Demand, Nitrogen compounds, Phos-
phorus, fecal coliform to name a few. The breakdown and
settling of these organics is accomplished in the biological/
secondary treatment mainly be micro-organisms called,
protozoans.
The biological/secondary treatment equipment gener-
ally consists of three components; treatment unit, settling
unit, sludge digester/holding unit. In the treatment unit the
sludge and preliminarily treated influent, are mixed with
dissolved oxygen for a specified amount of time. The sludge
consisting of micro-organisms, termed Mixed Liquor
Suspended Solids, (MLSS) metabolize and reproduce,
over time, when combined with dissolved oxygen. The
time needed, termed detention time, the MLSS require to
adequately metabolize the organic matter and reproduce
is dependent on the quantity and quality of MLSS popu-
lation and strength of the influent. To determine quantity
and quality of the MLSS, the operator must examine the
sludge under a microscope. After sufficient treatment is
achieved, the wastewater is transported via gravity or
pumps to another unit for settling. The settling unit allows
time with no agitation for the MLSS to settle to the bottom,
allowing clear wastewater to leave the tank and progress to
the chlorine contact tank. The MLSS may then be removed
from the bottom of the tank, with one portion “returned”
back to the treatment unit, and the other portion “wasted”
to sludge digestion/holding. The returned MLSS, again,
mixes with preliminarily treated influent and dissolved
oxygen to allow the micro-organisms time for metabolizing
the organic matter for reproduction. The wasted portion
goes to digestion and is not provided any organic matter,
only dissolved oxygen. In this way, the wasted sludge is
volatized by the dissolved oxygen and is considered stable
once the volatiles are reduced by 37%. The stable sludge
may be used on farm fields as a fertilizer and can be stored
in sludge holding. The tertiary treatment of the treated
wastewater from the settling tank consists of chlorination
and sand filtration.
The chlorination is designed to lower the bacterial
counts so the effluent does not harm the stream biota. Af-
ter chlorination the wastewater may be transported to sand
filtration where unsettled solids are removed. The unsettled
solids cause the effluent to be turbid. The turbidity is gen-
erally dead MLSS that did not settle in the settling tank.
The amount of turbidity can be reduced by adjusting the
return rate and the wasted rate of the MLSS in accordance
with the examination of the MLSS to determine the age,
diversity and quantity of the micro-organisms.
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Microscopic examination of the MLSS enables the op-
erator to determine the level of treatment and indicate set-
tling abilities in a matter of minutes. The microorganisms
within activated sludge “treat” the wastewater by reducing
the biochemical oxygen demand, organic and nitrogenous
demands. The most important of these microorganisms are
the heterotropic and autotrophic bacteria and the protozoa
in treatment and clarification of the wastewater.
The protozoa will clarify the wastewater in a tank that
provides “food” (the bacteria) and dissolved oxygen, the
aeration tank. With dissolved oxygen and food they will
reproduce to form a “floc particle”. Once the MLSS is
moved to a “settling, or Clarifier” tank, the floc particles
will be able to adhere to one another to form a “blanket” of
sludge. This blanket becomes heavier as it thickens which
allows it to “settle” to the bottom of the tank as the treated
and clarified water exits the settling tank from the top. It is
important to note that these protozoa must be returned to
the aeration tank before they die and float to the surface of
the settling tank.
The most important microorganisms for the operator to
have in the MLSS for clarification and setting are the proto-
zoa. As discussed previously, the protozoa eat the bacteria
and help to provide a clear effluent. There are three groups
of protozoa, each of which have significance in the treat-
ment of wastewater.
The three groups include the:
1. Amoeboids
2. Flagellates
3. Ciliates
Amoeboid
Amoeboid protozoa has extremely flexible membranes
that allow food to be absorbed through them. They move
throughout the MLSS by the movement of protoplasm
within the cell. Generally, Amoeboids are found high in
numbers in “young” MLSS. Generally, Amoeboids are
found high in numbers in “young” MLSS and is associ-
ated with poor settling sludge. Once the activated sludge is
allowed to age and exhibits good settling, the Amoeboids
population is about one sixteenth (1/16) of the total number
of important protozoans.
Flagellates
Flagellate protozoa move throughout the MLSS by the
corkscrew movement of the tail (Flagella) which extends
from their round or elliptical cell configuration. Generally,
Flagellates are found in high numbers where the Amoeboid
population is low, and a high organic load (BOD) exists.
If these protozoa predominate the MLSS the settling abil-
ity of the MLSS is poor and the clarity is only fair. As the
MLSS ages and exhibits good settling, the Flagellate popu-
lation is reduced to one-eighth (1/8) of the total number of
important protozoans.
Ciliates
These protozoa move throughout the MLSS by rotating
hair-like membrane (cilia) which cover all or part of their
cell membrane. Additionally, they use the cilia to pull food
into their gullet. There are two types of Ciliates, free swim-
ming and stalked Ciliates. Ciliates are associated with fair
to good settling of the activated sludge and comprise three
quarters (3/4) of the total number of important protozoans.
Free-Swimming Ciliates
Free-swimming ciliates are numerous when there are
large numbers of bacteria in the activated sludge. These
protozoa consume the bacteria thereby clarifying the efflu-
ent. The predominance of these ciliates indicates that the
wastewater treatment process is approaching an optimum
degree. In addition, their presence indicates that the set-
tling of the activated sludge will be increased.
Stalked Ciliates
Stalked Ciliates present in high numbers in the MLSS
indicate that the wastewater treatment has surpassed the
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reduction of carbonaceous biochemical oxygen demand
and is satisfying the nitrogenous oxygen demand. These
ciliates indicate that optimum treatment of the wastewater
is occurring . In addition, ciliates attach themselves to each
other along with suspended solids, forming large floc par-
ticles that settle well. Large numbers of Stalked Ciliates are
associated with “good settling sludge” and clear effluent
water above the settled sludge blanket.
In an ideal world, the operator should examine the
MLSS under the microscope on a daily basis, however for
monitoring purposes two or three times per week should be
sufficient. Additional examination should be performed if
the treatment or settling abilities of the wastewater treat-
ment plant appears to be compromised.
Each time the operator collects a sample of MLSS it
should be collected from the same well-mixed portion of
the aeration tank. In addition the operator should be fill
out a worksheet so that the abundance of organisms can be
correlated with treatment and settling.
Two other operational tools to compliment the micro-
scopic examination are the “30 minute Settling Test” and
calculation of the Food to Microorganism ratio.
The 30 minute settling test will confirm the microscopic
examination results by providing further information on
which group of microorganisms predominate the MLSS.
The operator must perform this test directly before, after or
concurrently with the microscopic examination to correlate
the results. A good settling Sludge will settle to fill half of
the Settleometer within 5 to 10 minutes. The blanket/water
interface should be even and the water above the blanket
should be very clear.
The food to microorganism ratio (f/m) can be computed
from Pounds (Lb) of Biochemical Oxygen Demand (BOD)
of the raw wastewater / Lbs of Volatile solids (MLSS x
Volatile solid%) in the aeration tank. The f/m ratio should
be kept around 0.2 - 0.5 to provide optimum treatment,
however every wastewater treatment system is different
and the operator should determine what ratio provides the
best treatment and settling. This test relies on the B.O.D.,
therefore the f/m result is 5 days old and can only be cor-
related with the 5 day old microscopic examination and 30
minute settling test worksheets. Once the operator calcu-
lates the f/m and compares it to the microorganisms and
settling, the operator can determine the correct amount of
MLSS to maintain in the aeration tank to provide optimum
treatment and clarity. Due to the fact the operator can not
generally control the strength of raw wastewater, the opera-
tor must adjust the amount of MLSS through returning the
correct amount back to the aeration tank from the settling
tank and removing or “wasting” any excess out of the sys-
tem to a digester or holding tank.

Needed additional equipment includes:

1. Slides
2. Coverslips
3. Several small dropping pipettes (disposable are most con-
veinent)
4. (Storage box
5. Dust cover.

Use of the Microscope

Procedures for preparing slides:
1. Clean cover slip and slide.
2. Use pipette to grab a sample of MLSS. Put finger on top of pi-
pette until the immersed end of a wide tip pipette reaches the
IV. Procedure bottom of sludge sample. Release your finger to allow sludge
into the pipette. Replace your finger on top of pipette and
Microscope Selection remove the pipette from the sampler beaker. A long tipped
Note: A relatively low cost microscope is suitable, how- eyedropper may also be used.
ever a dissecting microscope does NOT have the magnifica- 3. Place one drop of MLSS from the pipette to the middle of the
tion ability needed. glass slide.
Features that are desirable: 4. Pick up cover slip by two corners.
1. Built-in illumination or an external system which allows 5. Pull cover slip along glass slide towards drop of MLSS.
variations of light intensity. 6. As soon as cover slip touches drop of MLSS allow cover slip
2. A condenser system. to fall onto glass slide.
3. A movable stage. Stage should be controlled by coaxial 7. Pick up glass slide. Place on microscope stage.
handle rather than a manual push-pull. 8. Move stage up to within approximately 1/8 inch of objective
4. 10 X and 40 X objectives. on 10 X.
5. 10 X eyepiece. 9. Use the coarse then fine adjustments on the microscope to
bring the MLSS into the field of focus.
10. Turn to 45 X and repeat #9.
11. Identify organisms in the MLSS.

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 Procedures for Microscopic Examination
When performing a microscopic examination of MLSS,
the operator should fill out a worksheet that at a minimum
records:
1. Date and time of sample
2. Sample location
3. Name of operator
4. Type of microorganism
5. Approximate number within field view
6. size of floc particle (use 10 x to determine)

The operator should examine at a minimum three slides

per sample. Ideally the MLSS should be examined under
the microscope on a daily basis, however for monitoring
purposes twice to three times per week should be sufficient.
Additional examination should be performed if the treat-
ment or settling abilities of the wastewater treatment plant
appears to be compromised.
Food / Microorganism (F/M) Ratio
1. Lbs BOD/Lbs MLSS x VS% in aeration
2. Flow (MGD) x BOD (mg/L) x 8.34
( MLSS x %Volatile) x aeration tank volume x 8.34 x 7.48 / 1000000
NOTE: Good F/M ratio is around 0.2 - 0.5.

Sample Worksheet for Microscopic Analysis EXAMPLE:

1. Flow = 4.7 MGD
2. Raw wastewater BOD = 149 mg/L
3. 8.34Lb = weight of one gallon of water
4. %Volatile = 77%
5. MLSS = 1860 mg/L
6. 5 Tanks = 210’ x 20’ x 14’
7. 7.48 = gallons for 1 cubic foot
8. 1,000,000 gpd = 1 MGD

Calculation:
{4.7MGD x 149 mg/L x 8.34} = 5,840.502

0.77 x 1860 mg/L x [5(210 x 20 x 14) x 7.48 ] x [8.34 /

1,000,000] = 26,267.495

5,840.502 divided by 26,267.495 = .222

Conclusion:
Evaluation of Microscopic Examination as related to the
30 min. Settling test and F/M ratio

Procedure for 30 minute Settling test

Settleometer
1. Collect 1 Liter of MLSS from the same sampling site used for
the microscopic examination of MLSS above - a well mixed
portion of the aeration tank.
2. Pour the sample into a Settleometer and slowly mix for 15
seconds.
3. After setting the timer for 30 minutes, allow to set and record
the height of the settled sludge blanket at 5, 10, 20, and 30 min-
ute intervals. If the operator identifies the Amoeboid as the most
NOTE: Good settling sludge will settle to 500 mL, with clear liquid abundant, then the organic load in the aeration tank will be
above the sludge blanket within 5 to 10 minutes. high and the settling abilities of the MLSS will be very poor.

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The settling of this MLSS will be around 800 - 950 mL after The settling will be around 500 within 5 to 10 minutes
30 minutes. The F/M ratio will be close to or more than 1. and the F/M ratio will be around 0.5. Stalked Ciliates at-
tach to one another and to other matter causing floc par-
F/M = (5840.502 divided by 5267.495) = 1.1 ticles to form and grow. In the clarifier the floc particles are
able to adhere together forming a blanket that will settle to
Ideally the number of Amoeboid would be approxi- the bottom of the tank.
mately 1/16 of the total number of the three protozoa
discussed. Due to the fact that these organisms do not F/M =(5840.502 divided by 26,267.495) = .222
adhere well to one another or other protozoa they remain
suspended in the waster causing the clarity to be very poor.
To help rectify this situation the operator should allow as
much detention time in the aeration tank as possible to
facilitate additional microorganism growth.

If the operator identifies the flagellates as the most

abundant, the amoeboid population is low, and the organic
load (BOD) is high and the settling ability is poor resulting
in turbid water. The settling will be around 700 - 950 mL
after 15 minutes. The F/M ratio will be 0.8 or more.

F/M = (5840.502 divided by 7267.495) = 0.8

Ideally the number of flagellates needed to treat the

wastewater and exhibit good settling, the Flagellate popu-
lation should be reduced to one-eighth (1/8) of the total
number of important protozoans. Again, to help rectify
this situation the operator should allow as much detention
time in the aeration tank as possible to facilitate additional
microorganism growth. The most appropriate amount of Stalked Ciliates to
If the Ciliates,free swimming and stalked Ciliates are treat the wastewater and exhibit good settling is three quar-
identified as most abundant, then the B.O.D. will be less ters (3/4) of the total number of important protozoans. The
than 10 mg/L and the nitrogenous demand will be satisfied operator should monitor the situation to keep the balance
reducing the ammonia to 3 mg/L or less. of food to microorganism as it is now.

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