Module 2

Wastewater Treatment

CONTENTS

Parameter Equipment
Settling Velocity Settling Column Test
Jar test Jar flocculator
Chemical Oxygen Demand Spectrophotometer and Testing Kits
Biochemical Oxygen Demand Oxygen Meter
Flocculation Micro Sentinel
VFA Titration
Coliform Bacteriological analysis
 SETTLING COLUMN

Laboratory settling column tests can be used to:

Model the behavior of flocculent settling, evaluation of existing settling tanks, developing data
for plant expansion or modification. However, it is not practical for the design of new treatment
plants – not easy to estimate concentration of particles that will come from the
coagulation/flocculation units.

Settling column dimension: 150 mm in diameter to minimize side wall effects, height should be
at least equal to the proposed sedimentation tank .Sampling ports should be provided at equal
intervals in height

Samples should be removed from the ports at periodic time intervals, SS concentration should be
determined

Percent SS removal is calculated for each sample

Percent removal is plotted on a “time” versus “depth of collection” graph

Percent removal lines (R curves) are drawn by interpolation 𝑅% = 1 – (𝐶𝑡/ 𝐶𝑂 )∗ 100

 JAR TEST

Procedure for Laboratory Jar Test

The purpose of the laboratory jar test is to select and quantify a treatment program for removal of
suspended solids or oil from raw water or a dilute process or waste stream. Jar tests are conducted on a
four- or six-place gang stirrer, which can be utilized to simulate mixing and settling conditions in a
clarifier. Jars (beakers) with different treatment programs or the same product at different dosages are
run side-by-side, and the results compared to an untreated jar, or one treated with the current program.
The general procedure for jar testing is as follows

Coagulants example : Aluminum Sulfate (75-250 g/m3)

Ferric chloride (45-90 g/m3)
Fill the appropriate number of (matched) 1000 mL square transparent jars2 with well-mixed test
water, using a 1000 mL graduate. Place the filled jars on the gang stirrer, with the paddles positioned
identically in each beaker.
A. After the beakers have been filled to the 1,000 ml mark with the water to be tested, begin stirring the
water at 100 rpm or maximum speed on the gang stirrer.

B. Add the coagulant dosages to the beakers as previously determined; in the example

C. Allow the coagulant to mix at the rapid speed of 100 rpm for approximately 2 minutes, or try to
duplicate the amount of agitation and mix time provided by the existing plant treatment system. The
timeframe should be correlated with the retention time associated with the actual injection pint of the
coagulant in the current application. Generally, the turbulence and mixing achieved at the injection point
in the application is much greater than can be generated in a jar test. Square jars provide more turbulence
than round jars.

D. During this fast mix procedure, observe the jars very closely to determine which dosage yields the first
floc or formation of particles. Make note which dosage showed this characteristic.

E. As the 2-minute rapid mix time comes to an end, observe which dosage yields the largest floc size and
clarity of water.
F. After the 2-minute rapid mix time, reduce the speed to a slower mix of approximately 30-40 rpm.
Allow the jars to mix at this lower speed for approximately 3 minutes or the times correlating to the plant
system. During this period, continue to evaluate the floc size and clarity of water.

G. At the end of the slow mix, turn the stirrers off completely and allow the floc to settle to the bottom of
the jar, or possibly float, depending on the treatment application. Make note of which dosage yields the
most rapid settling or floating rate, largest floc particles, and the clarity of water. After the jars have set
for approximately 2 to 5 minutes, you can extract some water from the jars and run a turbidity analysis to
determine more accurately which jar yields the best clarity water.

You should add the flocculent at the end of the 2-minute rapid mix period, but prior
to reducing the speed to 40 rpm. After adding the flocculent at the high rapid mix speed, allow for an
additional 30 seconds to 1 minute mixing to ensure that the flocculent is completely
dispersed in the jar. Then reduce the speed to 30-40 ppm and continue to evaluate the water quality in the
jar using the same procedure stated above.

H. Evaluating Flocculent Chemistries. After the most effective coagulant has been determined, it is
sometimes beneficial to determine which flocculent will yield large, stable floc particles
and more rapid settling or flotation. Keep in mind that coagulant is the primary chemistry used for charge
neutralization or precipitation and to initiate the formation of floc particles. The flocculent chemistry is
used to increase the size of the floc particles produced and improve settle ability
 DOSAGE (mg/L) Floc
Test Floc Settling Supernatant Other COMMENTS
No. Size Rate Clarity

Signed Page No. of Pages

 CHEMICAL OXYGEN DEMAND

Using the THERMO Spectrophotometer and COD testing Kits

Introduction:

Chemical Oxygen Demand is a mean of measuring the pollution strength of wastewater. By

using this method, most oxidizable organic compounds present in the wastewater sample may be
measured: COD measurements are preferred when a mixed domestic-industrial waste is entering
a plant or where a more rapid determination of the load is desired.

The chemical oxygen demand test has a major advantage over the biochemical oxygen demand
analysis because of the short time required for performance – a few hours as opposed to five
days for the standard BOD test. Since this test can be run in several hours, it gives the operator a
more timely idea of what is entering the plant and how the plant is performing. This permits
closer operational control of the treatment process.

Generally, COD values are higher than BOD values. The reason is that biochemical oxygen
demand measures only the quantity of organic material capable of being oxidized, while the
chemical oxygen demand represents a more complete oxidation. Typical COD values for
domestic waste range from 200 – 500 mg/L.

Sample Collection

Collect samples in glass bottles. When it is necessary to preserve samples for storage, acidify to
pH < 2 with concentrated sulfuric acid. Store preserved samples at 4˚C for no longer than 28
days after collection.

Experimental Procedure:

1. Homogenize 500ml of sample for 2 minutes in the blender.

2. Preheat the digester block to 150˚C.
3. Remove the cap from a COD vial.
4. Pipet 2.00 ml (0.2 ml for 0-15,000 mg/L range) of sample into the vial. The contents of
the vial will become hot.
5. Replace the cap onto the COD vial, making certain that it is secured tightly.
6. Carefully invert the vial several times to mix the contents.
7. Wipe the vial with a damp towel and place it in the preheated digester block.
8. Prepare the reagent blank by repeating steps 3 through 7, using deionized water rather
than sample in step 4.
9. Allow the vials to heat in the digester block at 150˚C for 2 hours. (Countdown starts
immediately when the 150˚C is reached).
10. Turn the digester block off and allow the vials to remain in the unit to cool for 15 to 20
minutes.
11. Wipe the exterior of the reagent blank vial until it is clean and dry. Place the reagent the
zero concentration point.
12. Wipe the exterior of a test COD vial until it is clean and dry. Place the vial into the
instrument sample compartment to obtain test results.
 BIOCHEMICAL OXYGEN DEMAND

Introduction:

The BOD of wastewater is a common indicator of the fraction of organic matter that may be
degraded by microbial action at a given time period at a temperature of 20˚C. The test is related
to the oxygen that would be required to stabilize the waste after discharging to a receiving body
of water.

Experimental Procedure:

Source: Wastewater Engineering – Metcalf & Eddy

The most widely used parameter of organic pollution applies to both wastewater and surface
water is the 5-day BOD (𝐵𝑂𝐷5 ). This determination involves the measurements of the dissolved
oxygen used by microorganisms in the biochemical oxidation of organic matter. Despite the
widespread use of the BOD test, it has a number of limitations (which are discussed later in this
section). It is hoped that, through the continued efforts of workers in the field, one of the other
measures of organic content, or perhaps a new measure, will ultimately be used in its place.
Why, then, if the test suffers from serious limitations, is further space devoted to it in this text?
The reason is that BOD test results are now used (1) to determine the approximate quantity of
oxygen that will be required to biologically stabilize the organic matter present, (2) to determine
the size of waste treatment facilities, and (3) to measure the efficiency of some treatment
processes, and (4) to determine compliance with wastewater discharge permits. Because it is
likely that the BOD test will continue to be used for some time, it is important to know as much
as possible about the test and its limitations.

To ensure that meaningful results are obtained, the sample must be suitably diluted with a
specially prepared dilution water so that adequate nutrients and oxygen will be available during
the incubation period. Normally, several dilutions are prepared to cover the complete range of
possible values. The ranges of BOD that can be measured with various dilutions based on
percentage mixtures and direct pipetting are reported in table 1.

When the sample contains a large population of microorganisms (untreated wastewater, for
example), seeding is not necessary. If required, the dilution water is “seeded” with a bacterial
culture that has been acclimated to the organic matter or other materials that may be present in
the wastewater. The seed culture that is used to prepare the dilution water for the BOD test is a
mixed culture. Such cultures contain large numbers of saprophytic bacteria and other organisms
that oxidize non-carbonaceous matter. A variety of commercial seed preparations are also
available.

The incubation period is usually five days at 20˚C, but other lengths of time and temperatures
can be used. Longer time periods (typically seven days), which correspond to work schedules,
are often used, especially in small plants where laboratory staff is not available on the weekends.
The temperature, however, should be constant throughout the test.

Dilution Requirements

The BOD concentration in most wastewaters exceeds the concentration of dissolved oxygen
(DO) available in an air-saturated sample. Therefore, it is necessary to dilute the sample before
incubation to bring the oxygen demand and supply into appropriate balance. Because bacterial
growth requires nutrients such as nitrogen, phosphorus, and trace metals, these are added to the
dilution water, which is buffered to ensure that the pH of the incubated sample remains in a
range
suitable for bacterial growth. Complete stabilization of a sample may require a period of
incubation too long for practical purposes; therefore, 5 d has been accepted as the standard
incubation period.
If the dilution water is of poor quality, the BOD of the dilution water will appear as sample
BOD. This effect will be amplified by the dilution factor. A positive bias will result. The
methods included below (Section 5210B and Section 5210C) contain both a dilution-water
check and a dilution-water blank. Seeded dilution waters are checked further for acceptable
quality by measuring their consumption of oxygen from a known organic mixture, usually
glucose and glutamic acid.
The source of dilution water is not restricted and may be distilled, tap, or receiving stream
water free of biodegradable organics and bio inhibitory substances such as chlorine or heavy
metals. Distilled water may contain ammonia or volatile organics; deionized waters often are
contaminated with soluble organics leached from the resin bed. Use of copper-lined stills or
copper fittings attached to distilled water lines may produce water containing excessive amounts
of copper.

Reagents

Prepare reagents in advance but discard if there is any sign of precipitation or biological
growth in the stock bottles. Commercial equivalents of these reagents are acceptable and
different stock concentrations may be used if doses are adjusted proportionally.
a. Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g
Na2HPO47H2O, and 1.7 g NH4Cl in about 500 mL distilled water and dilute to 1 L. The pH
should be 7.2 without further adjustment. Alternatively, dissolve 42.5 g KH2PO4 or 54.3 g
K2HPO4 in about 700 mL distilled water. Adjust pH to 7.2 with 30% NaOH and dilute to 1 L.
b. Magnesium sulfate solution: Dissolve 22.5 g MgSO47H2O in distilled water and dilute to
1 L.
c. Calcium chloride solution: Dissolve 27.5 g CaCl2 in distilled water and dilute to 1 L.
d. Ferric chloride solution: Dissolve 0.25 g FeCl36H2O in distilled water and dilute to 1 L.
e. Acid and alkali solutions, 1N, for neutralization of caustic or acidic waste samples.
1) Acid—Slowly and while stirring, add 28 mL conc sulfuric acid to distilled water. Dilute
to 1 L.
2) Alkali—Dissolve 40 g sodium hydroxide in distilled water. Dilute to 1 L.
f. Sodium sulfite solution: Dissolve 1.575 g Na2SO3 in 1000 mL distilled water. This
solution is not stable; prepare daily.
g. Nitrification inhibitor, 2-chloro-6-(trichloromethyl) pyridine.*#(2)
h. Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade glutamic acid at
103°C for 1 h. Add 150 mg glucose and 150 mg glutamic acid to distilled water and dilute
to 1 L. Prepare fresh immediately before use.
i. Ammonium chloride solution: Dissolve 1.15 g NH4Cl in about 500 mL distilled water,
adjust pH to 7.2 with NaOH solution, and dilute to 1 L. Solution contains 0.3 𝑚𝑔 𝑁/𝑚𝐿.
j. Dilution water: Use demineralized, distilled, tap, or natural water for making sample
dilutions.

Sample pretreatment: Check pH of all samples before testing unless previous experience
indicates that pH is within the acceptable range.
1) Samples containing caustic alkalinity (pH >8.5) or acidity (pH <6.0)—Neutralize samples
to pH 6.5 to 7.5 with a solution of sulfuric acid (H2SO4) or sodium hydroxide (NaOH) of such
strength that the quantity of reagent does not dilute the sample by more than 0.5%. The pH of
dilution water should not be affected by the lowest sample dilution. Always seed samples that
have been pH-adjusted.

Dilution technique: Make several dilutions of sample that will result in a residual DO of at
least 1 mg/L and a DO uptake of at least 2 mg/L after a 5-d incubation. Five dilutions are
recommended unless experience with a particular sample shows that use of a smaller number of
dilutions produces at least two bottles giving acceptable minimum DO depletion and residual
limits. A more rapid analysis, such as COD, may be correlated approximately with BOD and
serve as a guide in selecting dilutions. In the absence of prior knowledge, use the following
dilutions: 0.0 to 1.0% for strong industrial wastes, 1 to 5% for raw and settled wastewater, 5 to
25% for biologically treated effluent, and 25 to 100% for polluted river waters.
Prepare dilutions either in graduated cylinders or volumetric glassware, and then transfer to
BOD bottles or prepare directly in BOD bottles. Either dilution method can be combined with
any DO measurement technique. The number of bottles to be prepared for each dilution depends
on the DO technique and the number of replicates desired.
When using graduated cylinders or volumetric flasks to prepare dilutions, and when seeding
is necessary, add seed either directly to dilution water or to individual cylinders or flasks before
dilution. Seeding of individual cylinders or flasks avoids a declining ratio of seed to sample as
increasing dilutions are made. When dilutions are prepared directly in BOD bottles and when
seeding is necessary, add seed directly to dilution water or directly to the BOD bottles. When a
bottle contains more than 67% of the sample after dilution, nutrients may be limited in the
diluted sample and subsequently reduce biological activity. In such samples, add the nutrient,
mineral, and buffer solutions directly to individual BOD bottles at a rate of 1
mL/L (0.33 mL/300-mL bottle) or use commercially prepared solutions designed to dose the
appropriate bottle size.
Determination of initial DO: If the sample contains materials that react rapidly with DO,
determine initial DO immediately after filling BOD bottle with diluted sample. If rapid initial
DO
uptake is insignificant, the time period between preparing dilution and measuring initial DO is
not critical but should not exceed 30 min.
Dilution water blank: Use a dilution water blank as a rough check on quality of unseeded
dilution water and cleanliness of incubation bottles. Together with each batch of samples
incubate a bottle of unseeded dilution water. Determine initial and final DO .The
DO uptake should not be more than 0.2 mg/L and preferably not more than 0.1 mg/L Discard all
dilution water having a DO uptake greater than 0.2 mg/L and either eliminate source of
contamination or select an alternate dilution water source.
Incubation: Incubate at 20°C ± 1°C BOD bottles containing desired dilutions, seed
controls, dilution water blanks, and glucose-glutamic acid checks.
Determination of final DO: After 5 d incubation determine DO in sample dilutions, blank.

Calculation
For each test bottle meeting the 2.0-mg/L minimum DO depletion and the 1.0-mg/L residual
DO, calculate BOD5 as follows:
When dilution water is not seeded:

𝑚𝑔 𝐷1 − 𝐷2
𝐵𝑂𝐷 = 𝐸𝑞. 1
𝐿 𝑃

where:
D1 = DO of diluted sample immediately after preparation, mg/L,
D2 = DO of diluted sample after 5 d incubation at 20°C, mg/L,

P = decimal volumetric fraction of sample used,

B1 = DO of seed control before incubation, mg/L (¶ 4d),
B2 = DO of seed control after incubation mg/L (¶ 4d), and
f = ratio of seed in diluted sample to seed in seed control = (% seed in diluted
sample)/(% seed in seed control).

Biochemical oxidation is a slow process and theoretically takes an infinite time to go to

completion. Within a 20-day period, the oxidation of the carbonaceous organic matter is about 95
to 99 % complete, and in the 5-day period used for the BOD test, oxidation is from 60 to 70 %
complete. The 20˚C temperature used is an average value for slow-moving streams in
temperature climates and is easily duplicated in an incubator. Different results would be obtained
at different temperatures because biochemical reaction rates are temperature dependent.
 FLOCCULATION

Using Micro Sentinel

Introduction:

Chemical flocculation is widely used to treat water containing suspended solids. Chemical are
added to encourage the particles of solid to clump together, usually by inhibiting the electrostatic
forces, which hold them apart. The standard approach to achieve this is to add a highly valent
(high charged) ionic material, usually an Iron or Aluminum Salt. Dispersed insoluble particles
will then tend to group together forming aggregates and separate from the water phase.
Depending on their density, these will either sink forming a sludge or float to the surface.
Materials with about the same density as water are more difficult to separate. WMEC Optimized
Flocculation includes treatment with Iron salts to destabilize suspended solids. Particularly
common types of hazardous contamination are the heavy metals (copper, lead, cadmium,
chromium etc.) and these occur in many industrial processes. Often they occur however in the
form of dissolved salts. In order to separate these, the alkalinity (pH) of the water needs to be
increased, ideally up to about pH 10, when most of these metals form insoluble hydroxides,
which Fall out of solution. WMEC Optimized Flocculation includes pH adjustment to drop out
most heavy metal hydroxides.

Similarly, in the course of pH adjustment, iron and aluminum salts also form insoluble
hydroxides. These are fluffy gel like solids suspension, and they have a very large surface area.
The surface of these hydroxide flocks is also very active and acts as an absorbent for many types
of dissolved molecules and dispersed liquids. Many dissolved contaminants are preferentially
absorbed onto the suspended hydroxide flocks. Many small suspended solid particles are also
trapped in the forming iron hydroxide gels and therefore are easily separated. WMEC Optimized
Flocculation causes many contaminants to be removed by adsorption on the hydroxide gels
created. The formation of hydroxides from both added flocculants and heavy metal salts present
does not always enable us to predict whether or not the separated solids will sink or float.

This will be influenced by the contaminants present:

• Absorbed oil and entrained air bubbles may cause solids to float
• Dense heavy metal sludge are more prone to sink.

A varying composition waste stream may in fact produce both sinking and floating solids with
the same chemical treatment. This may be managed by the addition of a dense solid, which
flocculated with the hydroxide flocks formed and forces them to sink. Specially selected clays
are ideal for this purpose and may absorb further remaining quantities of contaminants from
solution. WMEC Optimized Flocculation includes treatment with selected clay, which improves
absorption effectiveness and forces all the solids to sink rather than float.
The rate of settlement of the solids depends largely on the size of the flocks under any given
conditions. Increasing the size of the flocks shortens settlement times, improves the filtration
behavior of the flocks and speeds up the entire treatment process. This is achieved by the
addition of proprietary soluble “destabilizing polymer” coagulant, or polyelectrolytes. These are
used in minute concentration and are non-hazardous (used for treating drinking water). They
work by attaching themselves to the flocks and in fact are mostly removed with them. WMEC
Optimized Flocculation includes final treatment with polyelectrolyte coagulant. When all the
above processes are working well, a dense and easily filtered sludge of flocks results which
normally concentrates a high proportion of the contaminants into about 2-5% w/w of the original
waste stream. The disposal of the resulting small quantity of sludge is commercially available at
much lower cost than that of disposing of the entire volume of untreated contaminated water.

Summary

• Addition of high valent flocculants to destabilize suspended solids

• pH adjustment to precipitate heavy metals and form hydroxide flocks
• Clay treatment to promote flock sinking and improve adsorption
• Poly-electrolyte flocculation to accelerate settlement and improve filtration

When these steps are taken into account, the difference between the crude treatment with caustic
or polyelectrolyte and WMEC Optimized Flocculation becomes clear. The Sentinel range of
treatment plant is designed to manage all the above processes and provide an optimum match of
process engineering and chemistry. All the above steps result from use of the full Sentinel
treatment scheme, available either as four component kits or in bulk packs for the large user.

Experimental Procedure:

Stage 1: Filling and reagent addition:

1. Check the sludge transfer valve (V) is in the closed position.

2. Open the lid and fill the hopper with 201 of effluent (i.e. to bottom of the funnel lip).
3. Switch on the agitator with switch A.
4. Add the following amounts of reagent while stirring:
PACK 1 10g Stir for 5 minutes
PACK 2 30g Stir for 5 minutes
PACK 3 20g Stir for 5 minutes
PACK 4 5ml Stir for 5 minutes
5. Switch off agitator. The vessel contents should be colored and clear or very slightly
cloudy.
Stage 2: Liquid removal

6. Check that the sludge filter has a filter bag fitted.

7. Position the outlet hose in a suitable discharge or collection point.
8. Turn the sludge transfer valve (V) to the open position.
9. Start pump (P) and run until main vessel is empty.
10. Switch off pump and proceed to check sludge filter.

Note:

The liquid entering the first carbon filter will be colored.

The liquid in the second carbon filter bowl, which is transparent, should be colorless. If a reddish
color is visible, then the carbon filter 1 should be changed after discharge is complete.

Stage 3: Sludge removal:

The filter bag will normally need replacing after 1-3 batches of effluent have been treated. When
the bag appears to be full of sludge replace it as follows:

1. Ensure that the pump is run until the main vessel is empty. Close valve V and switch off
the pump, P.
2. Unlock the filter body housing and lift off. Pull off the filter bag complete with sludge
and place in a suitable wide necked container for waste disposal. This container should be
clearly marked as waste and set aside solely for this purpose.
It is recommended that gloves and safety glasses are worn while handling the filter bag.
 Procedures for Alkalinity and VFAs

Preparing stock solutions:

1. NaOH solution (0.1M): Put 4g of NaOH pellets in 1L of water. Mix well to dissolve the pellets
completely. Use a glass bottle (reaction is exothermal).
2. HCL solution (0.1M): Add 1 ml of concentrated HCL acid to 119 ml of de-ionized water

Preparing the sample:

1. Centrifugation: Centrifuge samples in conical tubes (5000 rpm; 10 min). Collect the supernatant (to
be used for alkalinity test).
2. Filtration: Place the filtering set with the 1.2 𝜇𝑚 filter. Apply vacuum and pour the supernatant of
each cylinder slowly.
When all the supernatant is filtered, turn off the vacuum pump, remove the filtering set and pour
the filtrate into a labeled beaker.

Calculations:

Total alkalinity: TA(mg/L) = V1(ml) x 100

Total VFA concentration = VFA(mg/L) = V3(ml) x 887

Experimental procedure
1. Prepare 25 ml of filtered sample.
2. Add 0.1M HCL until pH drops to 4
3. Boil the sample for 3 minutes
4. Back titrate with 0.1M NaOH to pH of 4 and 7
Experimental data:

Sample A:
Sample A
20ml (20g)
Titration with 0.1M HCl 1000 µl
to pH of 3.5
500 µl

200 µl

100 µl

50µl

Total Volume (ml) V1 =

Back Titration with 0.1M 1000 µl

NaOH to pH of 4
Boil it 500 µl

200 µl

100 µl

50µl

Total Volume (ml) V2 =

Back Titration with 0.1M 1000 µl

NaOH to pH of 7
500 µl

200 µl

100 µl

50µl

Total Volume (ml) V3 =

 BACTERIOLOGICAL ANALYSIS

Apparatus
Broth ampoule, M-COLIBLUE BROTH, ROSALIC BROTH
Sterile buffered dilution water 1
Membrane filter, 0.45 micron 1
Petri dish with absorbent pad, 47-mm 1
Filtration apparatus with aspirator or pump 1
Forceps, sterilized 1
Incubator 1

Sample collection
• Use a sterile glass or plastic container. Open the sample containers immediately before
collection and close immediately after collection. Do not put the lid or cap down. Do not touch
the lip or inner surfaces of the container.
• To collect a potable water sample from a faucet, spigot, hydrant or pump, let the
water flow at a moderate rate for 2–3 minutes. Remove the screens or aerators. Do
not use faucets or spigots that have a bad seal or that show a leak between
components.
• To collect a non-potable sample from a river, lake or reservoir, hold the container
below the water surface, then remove the cap. As an alternative, remove the cap and
push the container, mouth down, below the water surface to prevent the collection of
surface scum. Put the mouth of the container into the current. Fully fill the container
below the water surface. Collect a minimum of 100 mL of sample. Keep a minimum of 2.5 cm (1
inch) of airspace in the container.

Sample volumes
Use a sample volume that is applicable to the sample type. For samples with a low level
of bacteria such as finished, potable water, use 100 mL of sample. Use less sample for
non-potable water or water that contains more bacteria.
When the approximate bacteria level is unknown, analyze three different sample
volumes. Use the results from the sample volume that shows approximately 20 to
200 colonies for each membrane filter.

Sample dilution
Dilute samples that contain a high level of bacteria so that approximately 20 to
200 bacteria colonies grow on the membrane filter. Use the steps that follow to make
serial dilutions of the sample.
1. Wash hands thoroughly with soap and water.
2. Invert the sample container for 30 seconds (approximately 25 times).
3. Open a bottle of sterile buffered dilution water.
4. Use a sterile pipet to add 11 mL of sample into the dilution water bottle.
5. Put the cap on the dilution water bottle and invert for 30 seconds (25 times). This is a
10x dilution (sample is diluted by a factor of 10).
6. Add 11 mL of the 10-fold dilution to another dilution bottle (100x dilution). Mix well.
7. Add 11 mL of the 100-fold dilution to the third bottle (1000x dilution). Mix well.
8. If necessary, continue to dilute the sample.

Summary of method
Fecal coliform : M-FC with Rosolic Acid broth PourRite™ ampules (for fecal coliform)
A fecal coli form test is usually done on wastewater, river, bathing, and other non-potable
waters. Fecal coli forms that grow on the membrane form an acid that reacts with
an aniline dye in the medium. A blue color forms.
Use m-FC Broth with Rosolic Acid to increase specificity when high levels of non-coliform
bacteria can be in the sample, unless all of the organisms in the sample are stressed or
injured. The sample is poured through a membrane filter.
The membrane filter is moved to a Petri dish that contains a nutritional broth or agar. During
incubation, the bacteria grow and form colonies on the membrane filter. After incubation, the
filter is examined with a microscope for bacteria colonies.

The m-ColiBlue24 Broth is made so that coli forms other than E. coli form red colonies.
The percentage of red colonies is equivalent to the Coli forms, Total and E. coli,
m-ColiBlue24
Count all of the red and blue colonies as total coliforms. Count all of the blue colonies as
E. coli. Blue colonies can be blue to purple

M coli blue is used for total and E coli