PROJECT SEMESTER REPORT

B.Tech Biotechnology
(July-December, 2013)

Microbial Identification in Effluent Treatment Plant

And
Analysis of ETP

Submitted by
Sahib Bhanot
Roll No: 701000033

Under the Guidance of

Mr. R C Pantola

Department of Biotechnology & Environmental Sciences

Thapar University, Patiala
July, 2013

ACKNOWLEDGEMENT

I am highly indebted to Ind-Swift Laboratories Ltd. for imparting with those basics which are
building blocks for my future in Biotechnology. This report has been the outcomeof continuous
cooperation, effective guidance and support from all the people associated with it.
It is my earliest privilege to express my deep sense of gratitude and indebtedness to my major
advisor and training inchargeMs. Sarika (Manager-HR Department), at Ind-Swift
Laboratories Ltd., Derabassi for their expert and inspiring guidance, constructive criticism and
constant encouragement during the course of my training.
I extend my profound thanks to Mr. RC Pantola (Microbiology) at Ind-Swift Laboratories Ltd.,
Derabassi, who guided me in each and every step in my training period with continuous feedback
and insight, without which my report wouldnt have been possible.
I take this opportunity to express my sincere thanks to Mr. Sourabh for his inspiring and
valuable guidance.
Lastly I would like to thank all the executive and supporting staff at Ind Swift Laboratories Ltd.
and would also like to express my gratitude to Mr. Manoj Baranwal and Vasundhara mam,
and Thapar University for allowing me to persue my six months project training at Ind Swift
Laboratories Ltd.

INDEX

Industrial Profile..7
Summary..9
Introduction.10

ETP..10
Biochemical Tests..15
Standard Operating Procedures..23

Review of literature26
Experimental work done.........27

Detail process of ETP......27

Biochemical Tests........43
Analysis of ETP...55

Results and Discussions...63

References........74

List of figures

Collection Tank..28
Flash mixer and Flocculator...29
Primary tube settler29
Buffer Tank....30
Anaerobic Tank..31
SAFF Tank.31
Aeration Tank.32
Secondary tube settler.33
Treated water Tank.33
Carbon Filters.35
Dry sludge beds..36
Evaporation Tank...36

Industry Profile
One of the leading research driven pharmaceutical group of India having presence across 45
countries of the global with two listed entities IND-SWIFT LIMITED (manufactures of finished
dosage) and IND-SWIFT LABORATORIES LIMITED (Manufactures of Active Pharmaceutical
Ingredients and Advanced Intermediates) and one wholly owned subsidiary in USA.IND-SWIFT
is the second largest drug manufacturers of North India. Our multipurpose; multiplication
manufacturing set-ups are spread across the lush-green plains of northern India. Presently 30% of
the group turnover is contributed by exports.
IND-SWIFT is Chandigarh based pharmaceutical company, established in 1986 with a mission
of winning global customers through innovative pharmaceutical products. Three visionaries
Jains, Mehtas and Munjals, dedicated themselves to work for humanitys quest for longer,
happier and healthier lives.It has been promoted by IND-SWIFT LIIMITED in joint venture with
the Punjab State Industrial Development Corporation Limited (PSIDC). The various
establishment of the group are located in and around Chandigarh, in Haryana (Panchkula),
Himachal Pradesh (Parwanoo) and Punjab (Derabassi). IND-SWIFT is ISO 9001-2008, WHO
GMP certified and is listed on Bombay Stock Exchange and National Stock Exchange. INDSWIFT has been ranked 35th among top Indian Pharmaceutical companies. IND-SWIFT ensure
value for money and customer satisfaction globally, in doing so it have delivered long term
profitable returns to its investors, value to its partners and rewarding careers to the employees.
The facilities are built according to current guidelines of MHRA, EU, WHO, and accreditations
with ISO 14000.

The strength of the group is in its strategic and timely diversification, massive infrastructure,
7

physical resources and excellence. IND-SWIFT LABORATORIES Ltd. concentrated on the

manufacturing of Active Pharmaceutical Ingredient (API).
The company possess portfolio of 750 products with presence in high growth therapeutic
segments of Cardiology, Dialectology, Anti-depressant, anti-allergic, Anti- infective, Neurology
& Oncology with a nationwide distribution network. Apart from being among top Indian Pharma
companies, IND-SWIFT has also progressively embarked in diversification into multifarious
fields viz infrastructure, printing packaging and stationery, education, and media publication
with its every unit as an independent profit earning center.

IND-SWIFT GROUP

IND-SWIFT LIMITED
(FINISHED DOASGE)

IND-SWIFT
LABORATORIES LIMITED
(API)

PANCHKULLA
DERABASSI

SAMBA

PARWANOO

BADDI

Summary
Effluent Treatment Plant is an industrial structure designed to remove biological or chemical
waste products from water, thereby permitting the treated water to be used for other
purposes.Microbial Identification of ETP( Effluent Treatment Plant) includes the collection of
samples of Aerobic effluent and anaerobic effluent which are further grown on petri plates to
recognize the microorganisms present in the samples. For this we have to grow the colonies with
two different methods i.e Streaking and Pour Plating. It includes various Biochemical Tests for
the identification of microorganisms present in the samples. Biochemical Tests include various
tests like Catalase Test , Coagulase Test , Oxidase Test , IMViC Test.

There is preparation of different media which are used in microbial identification of ETP

Soybean Casein Digest Agar: It is a general purpose medium used for cultivation of a
wide variety of microorganisms and for sterility testing in pharmaceutical procedures

Sabouraud Dextrose Agar: Sabouraud Dextrose Agar is used for the cultivation of
yeasts, moulds and aciduric bacteria.

Further the analysis of ETP includes the understanding of working and principle of ETP and
going through various parts of it. In the end values of BOD , COD , TSS , TDS are calculated to
analyse these values , so that the water which is treated can be used for the irrigation and other
purposes.

INTRODUCTION
Effluent Treatment Plant
Industrial wastewater treatment covers the mechanisms and processes used to treat waters that have
been contaminated in some way by anthropogenic industrial or commercial activities prior to its
release into the environment or its re-use. Most industries produce some wet waste although recent
trends in the developed world have been to minimize such production or recycle such waste within
the production process. However, many industries remain dependent on processes that produce
wastewaters.
So, industries produce wastewater, otherwise known as effluent, as a bi-product of their production.
The effluent contains several pollutants, which can be removed with the help of an effluent
treatment plant (ETP). The clean water can then be safely discharged into the environment.

Industrial waste water sources

Food industry
Wastewater generated from agricultural and food operations has distinctive characteristics that set it
apart from common municipal wastewater managed by public or private sewage treatment plants
throughout the world: it is biodegradable and nontoxic, but that has high concentrations of
biochemical oxygen demand (BOD) and suspended solids (SS). The constituents of food and
agriculture wastewater are often complex to predict due to the differences in BOD and pH in
effluents from vegetable, fruit, and meat products and due to

The seasonal nature of food processing and post harvesting. Processing of food from raw materials
requires large volumes of high grade water. Vegetable washing generates waters with high loads of
particulate matter and some dissolved organics. It may also contain surfactants. Animal slaughter
and processing produces very strong organic waste from body fluids, such as blood, and gut
contents. This wastewater is frequently contaminated by significant levels of antibiotics and growth
hormones from the animals and by a variety of pesticides used to control external parasites.
Insecticide residues in fleeces is a particular problem in treating waters generated in wool
processing. Processing food for sale produces wastes generated from cooking which are often rich in

10

plant organic material and may also contain salt, flavorings, coloring material and acids or alkali.
Very significant quantities of oil or fats may also be present.
Complex organic chemicals industry
A range of industries manufacture or use complex organic chemicals. These include pesticides,
pharmaceuticals, paints and dyes, petro-chemicals, detergents, plastics, paper pollution, etc. Waste
waters can be contaminated by feed-stock materials, by-products, product material in soluble or
particulate form, washing and cleaning agents, solvents and added value products such as
plasticizers. Treatment facilities that do not need control of their effluent typically opt for a type of
aerobic treatment, i.e. Aerated Lagoons.

Treatment Methods
Effluent can be treated in a number of different ways depending on the level of treatment required.
These levels are known as preliminary, primary, secondary and tertiary (or advanced). The
mechanisms for treatment can be divided into three broad categories: physical, chemical and
biological, which all include a number of different processes (Table 1). Many of these processes will
be used together in a single treatment plant.

Treatment Level

Description

Process

Preliminary

Removal of large solids such as rags,

sticks, grit and grease that may
damage equipment or result in
operational problems

Physical

Primary

Removal of floating and settleable

materials such as suspended solids
and organic matter

Physical and chemical

Secondary

Removal of biodegradable organic

matter and suspended solids

Biological and
chemical

11

Tertiary/advanced

Removal of residual suspended

solids / dissolved solids

Physical, chemical and

biological

Wastewater Treatment Levels and Processes

Advantages of waste water systems

Stay in compliance
Reduce hauling and off-site treatment costs
Eliminate municipal fees
Reduce supply costs by recovering production materials out of the waste-stream for re-use
Eliminate unnecessary water usage during processing

Analysis of waste water

This includes COD, BOD , TSS, TDS.

Commonly used chemicals for analysis:

Coagulation and flocculation:- Acrylamide copolymers, aluminium chloride,

aluminiumsulphate,cationic polyacrylamide, diallyldimethyl ammonium/chloride
acrylamide copolymer, ferric chloride, ferric and ferrous sulphate, kaolinite, poly
(diallyldimethyl ammonium chloride), polyaluminium chloride, polyamines, starch,
polyethyleneamines, resin amines, sodium aluminate.

pH adjustment:- Calcium carbonate, calcium hydroxide, calcium oxide, carbon dioxide,

magnesium oxide, potassium hydroxide, sodium bicarbonate, sodium bisulfate, sodium
carbonate, sodium hydroxide, sulfuric acid/hydrochloric acid.

Disinfection and oxidation products:-Anhydrous ammonia, ammonium hydroxide,

calcium hypochlorite, chlorine, iodine, potassium permanganate, sodium chlorate,
sodium chlorite, sodium hypochlorite.

Algicide:- Copper sulphate, copper triethanolamine complexes.

12

Softening:- Calcium hydroxide, calcium oxide, sodium carbonate, sodium chloride.

Taste and odor control:-Activated carbon, chlorine, chlorine dioxide, copper sulphate,
ozone, potassium permanganate.

Dechlorinator and antioxidant:- Sodium metabisulfite, sodium sulfite, sulfur dioxide.

National Standards for waste water

Effluent from industries must meet the national effluent discharge quality standards set by the
Government. Consequently any ETP must be designed and operated in such a way that it treats the
wastewater to these standards.

The regulations state that these quality standards must be ensured from the moment of going into
trial production for industrial units. They also state that the Department of Environment can
undertake spot checks at any time and the pollution levels must not exceed these quality standards.
Furthermore, the quality standards may be enforced in a more stringent manner if considered
necessary in view of the environmental conditions of a particular situation.

The waste discharge quality standards differ according to the point of disposal. So, the standards are
different for inland surface water (ponds, tanks, water bodies, water holes, canals, river, springs or
estuaries); public sewers (any sewer connected with fully combined processing plant including
primary and secondary treatment); and irrigated land defined as an appropriately irrigated plantation
area of specified crops based on quantity and quality of wastewater.

13

Parameter

Inland

Public

Land for

surface

sewers

Irrigation

waters

Ammoniacal nitrogen

50

50

Arsenic

0.2

0.2

0.2

Biological oxygen demand

30

350

100

Boron

Cadmium

Chemical oxygen demand

250

Chlorides

1000

1000

600

0.1

2.0

Cyanides

0.2

0.2

Fluorides

15

Lead

0.1

1.0

Mercury

0.01

0.01

Nickel

Oil and grease

10

20

10

5.5-9.0

5.5-9.0

5.5-9.0

(for 5 days at 20 0 c)

Chromium (hexavalent)
Copper

Pesticides
pH
Phenolic compounds

14

Parameter

Inland

Public

Land for

surface

sewers

Irrigation

0.05

0.05

60

60

Sulphates

1000

1000

1000

Sulphides

Suspended solids

100

600

200

Total dissolved solids

2100

2100

2100

15

waters

Selenium
Sodium (%)

(inorganic)
Total residual chlorine
1
Zinc
5
Iron
_

BIOCHEMICAL TESTS:
A biochemical test refers to the chemical identification of unknown substances within a living
thing. The test quantitatively and qualitatively determines a particular substance like an enzyme
within the blood. A biochemical test can be used to diagnose a given disease.

15

There are various types of biochemical tests which are used to identify microorganisms which
are present within the effluent (industrial waste water).

These are as follows:

Catalase test
Coagulase test
Oxidase test
IMViC test
1. Indole test
2. Methyl red test
3. Voges-Proskauer test
4. Citrate utilization test

1. CATALASE TEST: This test is performed to understand the biochemical process of

hydrogen peroxide detoxification by aerobic bacteria through the production of the
enzyme catalase. It is helpful in describing how catalase production can be determined.
Principle:Some bacteria contain flavoproteins that reduce oxygen (O2), resulting in the
production of hydrogen peroxide (H2O2) and, in some cases, an extremely toxic superoxide (O2-).
Accumulation of these substances will result in death of the organism as they are powerful
oxidizing agents and destroy cellular constituents very rapidly unless they can be enzymatically
degraded. These substances are produced when aerobes, facultative anaerobes, and
microaerophiles use the aerobic respiratory pathway, in which oxygen is the final electron
acceptor, during degradation of carbohydrates for energy production.
A bacterium must be able to protect itself against such O2 products or it will be killed. Many
bacteria possess enzymes that afford protection against toxic O2 products. Facultative anaerobes
and obligate aerobes usually contain the enzymes superoxide dismutase, which has the ability
to catalyze the destruction of superoxide, and either catalase or peroxidase, which catalyze the
destruction of hydrogen peroxide as follows:

16

The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase may
explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the
toxic concentration of H2O2 cannot be degraded when these organisms are cultivated in the
presence of oxygen.
Organisms capable of producing catalase rapidly degrade hydrogen peroxide which is a
tetramer containing four polypeptide chains, which are usually 500 amino acids long. It also
contains four porphyrinheme groups (ie. iron groups) that will allow the enzyme to react with the
hydrogen peroxide. The enzyme catalase is present in most cytochrome-containing aerobic and
facultative anaerobic bacteria. Catalase is the enzyme which has one of the highest turnover
numbers compared to all other enzymes; one molecule of catalase has the ability to convert
millions of molecules of hydrogen peroxide to water and oxygen in each second.
Catalase production and activity can be detected by adding the substrate H2O2 to an appropriately
incubated (18- to 24-hour) tryptic soy agar slant culture. Organisms which produce the enzyme
break down the hydrogen peroxide, and the resulting O2 production produces bubbles in the
reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the
catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water and are
catalase negative.

17

Catalase activity is very useful in differentiating between groups of bacteria. For example, the
morphologically similar Enterococcus (catalase negative) and Staphylococcus (catalase positive)
can be differentiated using the catalase test.

2. COAGULASE TEST
This test is to understand the biochemistry of the enzyme coagulase, to explain how coagulase
confer a survival advantage to bacteria that produce this enzyme.
Principle: Coagulases are enzymes that clot blood plasma by a mechanism that is similar to
normal clotting. The coagulase test identifies whether an organism produces this exoenzyme.
This enzyme clots the plasma component of blood. The only significant disease causing bacteria
of humans that produce coagulase enzyme are Staphylococcus aureus.
Thus this enzyme is a good indicator of the pathogenic potential of S. aureus.
In human host, the action of coagulase enzyme produces clotting of the plasma by converting
fibrinogen to fibrin in the immediate vicinity of the bacterium as a means of protection by itself.
The fibrin meshwork that is formed by this conversion surrounds the bacterial cells or infected
tissues, protecting the organism from non-specific host resistance mechanisms such as
phagocytosis and the anti staphylococcal activity of normal serum. This enables the bacterium to
persist in the presence of a host immune response, which can lead to the establishment of
infection. Thus, coagulase is described as a virulence factor( disease- causing factor) of
Staphylococcus aureus. Citrate and EDTA are usually added to act as anticoagulants and
prevent false-positive results.
Most strains of S.aureus produce one or two types of coagulase; free coagulase and bound
coagulase. Free coagulase is an extracellular enzyme which reacts with prothrombin and its
derivatives. Bound coagulase is localized on the surface of the cell wall and reacts with - and chains of the plasma fibrinogens to form a coagulate. Free coagulase is an enzyme that is
secreted extracellularly and bound coagulase is a cell wall associated protein. Free coagulase

18

can be detected in tube coagulase test and bound coagulase can be detected in slide coagulase
test.
Slide coagulase test may be used to screen isolates of S.aureus and tube coagulase may be used
for further confirmation. There are seven antigenic types of free coagulase, but only one
antigenic type of bound coagulase exists. Free coagulase is always heat labile while bound
coagulase is heat stable.
In the test, the sample is added to rabbit plasma and held at 37 C for a specified period of time.
Clot formation occurs within 4 hours is interpreted as a positive result and indicative of a virulent
Staphylococcus aureusstrain. The absence of coagulation after 24 hours of incubation is a
negative result, indicative of an avirulent strain.

3. Oxidase test:
The oxidase test identifies organisms that produce the enzyme cytochrome oxidase by Gramnegative bacteria. Cytochrome oxidase involves in the electron transport chain by transferring
electrons from a donor molecule to oxygen. It is a hallmark test for the Neiserria. It is also used
to discriminate between aerobic Gram-negative organisms like Pseudomonas aeruginosa and
other Enterobacteriaciae.
The test can be carried out simply by swabbing some of the test culture into one of the boxes on
an oxidase dry slide. If there is a colour change of purple or blue at 30seconds 1 minute; then
the result is positive
Principle:The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an
enzyme of the bacterial electron transport chain.
When present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine)
to (indophenols) purple color end product. When the enzyme is not present, the reagent remains
reduced and is colorless.

Note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron
19

acceptor in respiration. This does NOT mean that they are strict aerobes. Bacteria that are
oxidase-negative may be anaerobic, aerobic, or facultative; the oxidase negative result just means
that these organisms do not have the cytochrome c oxidase that oxidizes the test reagent. They
may respire using other oxidases in electron transport.

4. IMViC TEST:
INDOLE TEST: The indole test screens for the ability of an organism to degrade the
amino acid tryptophan and produce indole. It is used as part of the IMViC procedures, a
battery of tests designed to distinguish among members of the family Enterobacteriaceae.

Theory:Tryptophan is an amino acid that can undergo deamination and hydrolysis by

bacteriathat express tryptophanase enzyme.

tryptophan + water = indole + pyruvic acid + ammonia

The chief requirement for culturing an organism prior to performing the indole test is that
the medium contains a sufficient quantity of tryptophan (5). The presence of indole when
a microbe is grown in a medium rich in tryptophan demonstrates that an organism has the
capacity to degrade tryptophan. Detection of indole, a by-product of tryptophan
metabolism, relies upon the chemical reaction between indole and pdimethylaminobenzaldehyde (DMAB) under acidic conditions to produce the red dye
rosindole (5, 8).

METHYL RED TEST: This test determines whether the microbe performsmixed acids
fermentationwhen suppliedglucose. Mixed acids fermentation results in accumulation of
a variety of acids and a significant drop in the pHof the medium.

Principle: The methyl red test is the "M" portion of the four IMViC tests used to
characterize enteric bacteria. The methyl red test is used to identify enteric bacteria based
20

on their pattern of glucose metabolism. All enterics initially produce pyruvic acid from
glucose metabolism. Some enteric subsequently use the mixed acid pathway to
metabolize pyruvic acid to other acids, such as lactic, acetic, and formic acids. These
bacteria are called methyl-red positive and include Escherichia coli and Proteus vulgaris.
Other enterics subsequently use the butylene glycol pathway to metabolize pyruvic acid
to neutral end-products. These bacteria are called methyl-red-negative and include
Serratiamarcescens and Enterobacteraerogenes

VOGES-PROSKAUER TEST: To differentiate among the enteric organisms such as

Escherichia coli, Enterobacteraerogenes and Klebsiellapneumoniae.
Principle: Differentiation of the principal groups of Enterobacteriaceae can be
accomplished on the basis of their biochemical properties and enzymatic reactions in the
presence of specific substrates. The IMViC series of tests (indole, methyl red, vogesproskauer, and citrate utilization) can be used. The enteric organisms are subdivided as
lactose fermenters and no fermenters. Escherichia coli, Enterobacteraerogenes,
Klebsiellapneumoniaeare

lactose

fermenters.

Salmonella

typhimurium,

Shigelladysenteriae, Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenesfaecalisetc

are non-lactose fermenters. The Voges-Proskauer (VP) test has found wide acceptance in
clinical laboratories as a means of classifying strains of Enterobacteriaceae, based on
acetone production.
The Voges-Proskauer test determines the capability of some organisms to produce non
acidic or neutral end products, such as acetyl methyl carbinol, from the organic acids that
result from glucose metabolism.The reagent used in this test is Barritt's reagent, consists
of a mixture of alcoholic a-naphthol and 40% potassium hydroxide solution. Detection of
acetyl methyl carbinol requires this end product to be oxidized to a diacety compound.
This reaction will occur in the presence of the a-naphthol catalyst and a guanidine group
that is present in the peptone of the MR-VP medium. As a result, a pink complex is
formed, imparting a rose color to the medium. Development of a deep rose color in the
21

culture 15 minutes following the addition of Barritt's reagent is indicative of the presence
of acetyl methyl carbinol and represents a positive result. The absence of rose color is a
negative result.

Actyl methyl carbinol

Alpha naphthol
diacetyl
Guanidine group of peptone (pink complex)

CITRATE UTILIZATION: The citrate test detects the ability of an organism to use
citrate as the sole source of carbon and energy
Principle: Citrate utilization test is commonly employed as part of a group of tests, the
IMViC (Indole, Methyl Red, VP and Citrate) tests, that distinguish between members of
the Enterobacteriaceae family based on their metabolic by-products. Citrate utilization
test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon
source and inorganic(NH4H2PO4) is the sole fixed nitrogen source.
When an organic acid such as citrate (Remember Krebs cycle) is used as a carbon and
energy source, alkaline carbonates and bicarbonates are produced ultimately. In addition,
ammonium hydroxide is produced when the ammonium salts in the medium are used as
22

the sole nitrogen source. The colour change of the indicator is due to alkali production by
the test organism as it grows on the medium. Growth usually results in the bromothymol
blue indicator, turning from green to blue. The bromothymol blue pH indicator is a deep
forest green at neutral pH. With an increase in medium pH to above 7.6, bromothymol
blue changes to blue.

There are many procedures which are mandatory to follow before enterance and before
the start of any experiment in Microbiology laboratory at ISLL.

STANDARD OPERATING PROCEDURES

1. Cleaning of Microbiology lab

PROCEDURE:

The waste material present in the dustbin shall be removed

The floor, workbench, and instruments shall be mopped with dry cloth

The floor shall be mopped with cloth wetted with disinfectant

The dilution ratio( percentage v/v diluents sterile purified water) shall be used
as per table.
S.no

Name of disinfectant

Recommende potency
(percentage v/v)

Dettol

Savlon

Germitol

Triple 256

Combtan DS

23

Steri AFM multi

1.5

Phenyl

2.5

The different disinfectant shall be used on alternate days

The disinfectants prepared shall be used for one day only and shall be
discarded after use

The record of cleaning shall be recorded as per format

2. Entry and Exit in Microbiology lab

PROCEDURE:

Entry and Exit in Microbiology lab should be documented with time and date

At the time of entering Microbiology lab remove all jewellery, shoes in

change room

Dip the hands and feet in disinfectant solution kept in change room 1

Now enter to change room 2

Take the set of sterile garments kept in garment cubicle without allowing them
to come in contact wih floor or walls

Put on the garments in following sequence

Nose mask

Head cover

Shoe cover

Ensure that the head cover in completely covered

Then put on the pair of gloves

Dont touch the center surface of the gloves with finger and palm

Spray 70% isopropyalcohol over palm and hand

24

3. Cleaning of Microbiological Glassware

PROCEDURE:

Collect all the glassware to be cleaned and sterilized

Add detergents like Extran which is suface active and should be used as per
label instructions

Soak the glassware as it is in soap solution for about 1 hour

Scrub all the glassware by brush in such a way that all remaining from inner
surface are washed out

Clean with plenty of water

Pour fresh soap solution

Rinse thoroughly with soap solution then with plenty of water

Place the glassware in inverted position on SS trays to drain out excess water

Dry the glassware in oven at 80-100 degree

The glassware cleaned and dried as above shall be used

25

Review of literature:

The Common Effluent Treatment Plant located in Kagal Five Star MIDC, Kagal is
implemented by SMS infrastructure, Nagpur. The present study gives the details of
CETP. Inlet and outlet sample for various parameters is analyzed. Parameters are
analyzed like pH, Total Suspended Solids (TSS), Total Dissolved Solids (TDS), oil and
grease, Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD). Kagal
MIDC has developed site near CETP where treated effluent from CETP is discharged by
High Rate Transportation System.

Managing and Monitoring Effluent Treatment Plants this Project is funded by the
Department for International Development, UK under its Knowledge and Research
Programme and the European Commission, under its Asia Pro Eco Programme. The
project is also undertaken in collaboration with the pollution component of the
Investment Support to MACH (MACH refers to the Managing Aquatic Ecosystems
through Community Husbandry project) which is funded by the Government of
Bangladesh. The work has been undertaken by the Stockholm Environment Institute, the
Bangladesh Centre for Advanced Studies and the University of Leeds.

Dr. Cundell in US has discussed the overall strategies that may be successfully applied to
microbial identification insupport of microbial monitoring ofutilities, pharmaceutical
ingredients,the manufacturing environment andfinished products. Emphasis has been
givento the justification of the microbialidentification program, selection ofidentification
methods and use of speciation in successful product failure investigations.

26

Experimental work (Methods and techniques)

Detail of ETP process

27

1. Primary Treatment:
Primary treatment is used for the removal of suspended solids, removal/collection of oil,
equalization of various effluents, pH adjustment, oxygen transfer, digestion of effluent etc.

Collection Tank:

Effluent coming from different production plants and other department of industry vary in pH,
COD, TDS, BOD, color etc. In collection tank; effluents with different pH, low COD, BOD,
TDS, high COD, BOD, and TDS comes and get equalized.The basic use of this tank is
equalization.

Flash Mixer and Flocculator:

After the water passes through the collection tank, it enters flash mixer. Here various chemicals
such as Alum, Caustic and Polyelectrolyte are added and the water is mixed rapidly in container.
The alum reacts with the dirt in the water to form a floc(coagulated sediment).These floc
particles trap the dirt present in the raw water. After the water passes through the flash mixer, it
28

then flows into the flocculation chamber. The water is mixed at a very slow speed by large
paddles.
This allows the delicate floc particles to grow in size like a snowflake on its way to earth. As the
floc grows, it traps additional dirt and suspended material. Vrious dosing pumps are set up to
provide constant flow rate of the chemicals as shown.

Primary Tube settler:

Its for the settling of sludge. Here a V-notch is provided so that there are minimum chances of
sludge particle to get carried away with clear liquor. The rate is also controlled. There are
parallel tubes inside it so that the holding time of the effluent increase and the contamination gets
more time to get settled. The settled sludge is sent to sludge drying bed and then after it is dried,
it is incinerated. Clear liquor is transferred further.

29

2. Secondary Treatment:
Desired quality in the primary treatment established, the effluent treatment will vary in the
secondary treatment based on the effluent characterstics. The degree of the treatment and post
treatment method, disposal method, etc. Plays a virtual role of deciding the treatment.

Buffer Tank:

It is just a holding tank to mantain constant flow of effluent. In this tank, effluent from septic
tank is also added.
The use of doing this is; the septic effluent contains nitrogen, phosphorous, bacteria and other
natural media needed for anaerobic digestion. So this step reduces the cost of media used in
anaerobic tank.

Anaerobic Tank:

This is for anaerobic digestion of organic matter and other biodegradable components of
anaerobic microorganisms. Microorganisms consume oxygen from the organic compounds and
hence BOD is reduced.
Sludge concentration is monitored and if it is more than a certain level, then it is removed from
the drain pipe provided at different heights. There are acid forming and methanogenic bacteria
which produces carbonic acid and methane. Methane produced is used as biogas.

30

SAFF Tank

SAFF is submerged Aerated Fixed Film. Corrugated fixed films are kept filled in the SAFF tank.
These have biomass (Biosludge) attached with them. The air is introduced through diffusers at
the bottom of the tank.
This air helps in keeping the inlet effluent mixed with whole liquor in the tank and provides
oxygen to dissolnve in the effluent. Sludge volume is maintained. If it is less, air is increased.
Outlet is from bottom and enters aeration tank is middle by gravity.

Aeration Tank

This process is exchange of gases between the water and atmosphere. The treatment method is
basically for the transfer of oxygen to water for the expulsion of carbon dioxide, hydrogen
sulphide, and other volstile substances.
31

It has very important role in the effluent/wastewater treatment applications include the
precipitation of the impurities like iron, manganese in certain forms, and reducing COD and
BOD. Aeration technologies consist of different type of aerators like Floating aerators, diffused
aerators, spray aerators etc. These aerators are used foe utilising the maximum kinetic energy.
Floating aerators are capable to treat the effluent with high velocity of specified applications and
needs, here diffused aerators are used.

Secondary Tube Settler

Its design and function is same as of primary tube settler. Polyelectrolyte is added in inlet
effluent in order to enhance the settling and removal of suspended solids in the incoming effluent
of secondary tube settler. The sludge at the bottom of secondary tube settler is circulated back
continuously into aeration tank, and if sludge volume is to be increased, then it is circulated into
SAFF also.

32

3. Tertiary Treatment
Tertiary treatment plants are basically the final stages of treatment of effluents. The method of
treatment largely depends upon the final requirement of treated effluent water quality. After the
tertiary treatment, the treated effluent can be safely discharged, reused or even taken into the
process directly.

Treated water tank

The clear liquor from secondary tube settler goes to the treated water tank. It is just the holding
tank and the level is maintained here.

33

Filtration system

Filtration is the process of removal of suspended solids, precipitated ions, removing turbidity etc.
Filters are designed for passing water usually downward, through a media where the suspended
solids get trapped and removed.
Different types of filter media are used to remove suspended solids; the most common is sand,
but anthracite is also widely used. When a coarse layer of very light weight material like
anthracite is placed on the top of the sand in a normal down flow filter is used, the coarse
suspended particle gets filtered in the anthracite layer and the finer particles in the sand layer,
resulting in a less turbid water. These two types of filteration leads to classification of filteration
into surface filteration as in the former case and in-depth filteration as in the latter case.

The filtration systems offered by ISLL are:

Sand filters

Different grade of sand with definite size are the specialty of these filters. These are vertical and
horizontal type with specialized design if distribution and collection systems to ensure proper
functioning of the system and operate with maximum efficiency. Coarse, quarts sands are used
for this and some cases Garnet is also used as the media of filteration.

Activated Carbon Filters

These filters technologies are used for the removal of chlorine and microorganisms theough
adsorption. Definite grade of high quality activated carbon with specialized design of distribution
and collection systems will offer maximum efficiency of the removal of chlorine and ensuring
long life in the carbon.
Activated

carbon

filteration

is

most

effective

in

removing

organic

contaminants;chlorineetc.because organics are often responsible for taste, odor, and color
problems. Activated Carbon filteration can generally be used to improve aesthetically objection
water.

34

Back Wash Recovery Plants

The cleaning operation of filter bed by reverse flow of water is called backwash. During
backwash the water enters from bottom of filters and passes through filter bed and loosens the
compact bed during service cycle and the suspended solids and other suspended impurities
comes out through the outlet. These backwash wastes have high suspended solids content. The
water shall be treated before discharge to avoid environmental problems. Normally water
consumption through this process is very large and backwash water recovery systems are capable
to treat the backwash waste and ensur complete reuse of the water back to the process with
acceptable TSS/Turbidity level. These systems are cost effective methods of treating the
backwash waste.

Sludge Drying Bed:

Dewatering aims to reduce the water content further so that the solids content of the sludge is
about 20%(equivalent to 1Kg dry sludge with 4L of water). The sludge can then be handeled like
a solid. Dewatering can be done mechanically using a filter press(employing pressure or
vaccum), or a centrifuge. It can also be done using drying beds.
A drying bed consists of 30cm bed of sand with under drainage. Sludge is applied on the sand
bed and is allowed to dry by evaporation and drainage of excess water over a period of several
weeks depending on climatic conditions. Bacterial decomposition of the sludge takes place
during process while moisture content is sufficiently high. During the rainy season the process

35

may take a longer time to complete, and sizing the area of drying beds should takr this into
account.

Spray Ponds/Evaporation Ponds:

High TDS effluent is collected in these tanks and it gets evaporated in air. Sometimes if some
problem comes in the plant, effluent is transferred in these tanks.

Procedure for Microbial Identification in Effluent treatment plant.

Collection of samples: The samples from effluent treatment plant were collected from
both anaerobic and aerobic tank for the identification.

36

Preparation of the media:Two different media which wererecommended for general

laboratory use were selected. The medium supported growth of many fastidious
organisms without the addition of serum.The two medium were:
1. Soybean Casein Digest Agar
2. Sabouraud Dextrose Agar.

1. Soybean Casein Digest Agar: It is a general purpose medium used for

cultivation of a wide variety of microorganisms and for sterility testing in
pharmaceutical procedures.
Composition:
Ingredients
Pancreatic digest of casein
Papaic digest of soyabean meal
Sodium chloride
Agar
Final pH ( at 25C)

Gms / Litre
15.000
5.000
5.000
15.000
7.30.2

Directions: Suspend 40 grams in 1000 ml distilled water. Heat to boiling to

dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure
(121C) for 15 minutes. Mix well and pour into sterile Petri plates.
Principle:Soyabean Casein Digest Agar is a widely used medium, which
supports the growth of wide variety of organisms even that of fastidious ones
such as

Neisseria, Listeria , and Brucella etc. The medium with addition of

blood provides perfectly defined haemolysis zones, while preventing the lysis of
erythrocytes due to its sodium chloride content. It has been frequently used in the
health industry to produce antigens, toxins etc. Its simple and inhibitor-free
composition makes it suitable for the detection of antimicrobial agents in the

37

food and other products. Tryptone Soya Agar is recommended by various

pharmacopoeias as sterility testing medium.
Tryptone Soya Agar conforms as per USP and is used in microbial limit test and
antimicrobial preservative - effective test. The combination of Pancreatic digest
of casein and papaic digest of soyabean meal makes this media nutritious by
providing amino acids and long chain peptides for the growth of microorganisms.
Sodium chloride maintains the osmotic balance.
Quality Control
Appearance: Cream to yellow homogeneous free flowing powder
Gelling: Firm, comparable with 1.5% Agar gel
Colour and Clarity: BasalMedium: Light yellow coloured clear to slightly
opalescent gel. pH of 4.0% w/v aqueous solution at 25C
pH: 7.10-7.50
Cultural response:Cultural characteristics was observed after an incubation for
Bacterial at 35-37C 18-24 hours and for Fungal at 20-25C <=5days.

2. Sabouraud Dextrose Agar: Sabouraud Dextrose Agar is used for the cultivation
of yeasts, moulds and aciduric bacteria.

Composition:
Ingredients
Dextrose

Gms / Litre
40.000

Mycological, peptone
Agar
Final pH ( at 25C)

10.000
15.000
5.60.2

Directions: Suspend 65 grams in 1000 ml distilled water. Heat to boiling to

dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure
(121C) for 15 minutes.
38

Principle:Sabouraud Dextrose Agar is Carliers modification of the formulation

described by Sabouraud for the cultivation of fungi (yeasts, moulds), particularly
useful for the fungi associated with skin infections. This medium is also
employed to determine microbial contamination in food, cosmetics, and clinical
specimens.
Mycological peptone provides nitrogenous compounds. Dextrose provides an
energy source. High dextrose concentration and low pH favours fungal growth
and inhibits contaminating bacteria from test samples Some pathogenic fungi
may produce infective spores which are easily dispersed in air, so examination
should be carried out in safety cabinet. For heavily contaminated samples, the
plate must be supplemented with inhibitory agents for inhibiting bacterial growth
with lower pH.
Quality Control:
Appearance: Cream to yellow homogeneous free flowing powder
Gelling: Firm, comparable with 1.5% Agar gel
Colour and Clarity of prepared medium: Light amber coloured clear to
slightly opalescent gel forms in Petri plates Reaction Reaction of 6.5% w/v
aqueous solution at 25C .
pH: 5.40-5.80
Cultural Response: Growth Promotion was carried out in accordance with the
(USP/EP/BP/JP), after an incubation at 20-25 C for 24-48 hours.Recovery rate
is considered as 100% for bacteria growth on Soybean Casein Digest Agar and
fungus growth on Sabouraud Dextrose Agar
Growth Promotion Test: Growth Promotion was carried out in accordance with
the harmonized method of ICH (USP/EP/BP/JP), after an incubation at 30-35 C
for 24-48 hours.Recovery rate is considered as 100% for bacteria growth on
Soybean Casein Digest Agar and fungus growth on Sabouraud Dextrose Agar.
39

Isolation of sample: On media was done using pour plate techniques and streaking for
both qualitative and quantitative analysis.

1. The aerobic sample was isolated on TSA medium (caseinsoyabean digest Agar).

Growth pattern on the media after incubation

There were different colonies found on the media with different physical properties.These
colonies were sub-cultured using streaking technique to obtain the pure cultures.Isolated pure
culture were found.
These are as follow with their physical properties:

The appearance of first pure culture (AP 1) was round and resembles that of a sphere
(cocci). Because of the way the bacteria divide and multiply, it appeared in clusters or
tetrads. It appeared as a large white to golden colony.

40

The appearance of second pure culture( AP2) of aerobic sample on TSA media was
pearlescent type. It was a rough appearance with mock orange color. Adiffusible pigment
of green blue color was seen on media.

The appearance of third pure culture( AP3) hasd little round structures bigger than that of
AP1. The appearance was a little opaque than AP 1. White to cream pigmented colonies
are found.

41

2. The anaerobic samples were isolated on TSA medium and the growth was checked on it
and the two different colonies in morphology were found. These colonies were further
sub cultured using streaking method and pure culture were obtained by this method.

The first sample (ANP1) isolated was consisting of straight rods having viscous/mucoid
appearance. The colonies formed were thick and appearing turbid white in color.

The appearance of second pure culture( ANP2) had little round structures bigger than that
of AP1. The appearance was a little opaque than AP1. White to cream pigmented
colonies are found. It was similar to AP3 Plate.

42

Biochemical Tests:Different Biochemical Tests were performed on these pure culture

to identify the microorganism present in them by comparing them with the standard
results.

Catalase Test:
Material Required:
Cultures: 24-48 hour tryptic soy broth cultures of bacteria

Media: Tryptic soy agar

Reagent: 3% hydrogen peroxide

(Storage:-Upon receipt, store at 2-8C away from direct light. Reagents should not be
used if there are signs of deterioration or if the expiration date has passed.)

Equipments:Bunsen burner, Inoculating loop, Test tubes, Test tube rack, Microscopic
slides.

Procedure:
The test can be done by two methods.
a) Slant method
b) Slide method

Slant Method:
Using a sterile technique, the organisms were inoculated using a labeled tube by means of
a streak inoculation.

Cultures were incubated for 24-48 hours at 37C.

Three or four drops of the 3% hydrogen peroxide were flown over the entire surface of
each slant culture.

Each culture was examined for the presence or absence of bubbling or foaming.

Slide Method:
Glass slide was divided into two sections with grease pencil. One was labeled as test
and the other as control.

Small drop of normal saline was placed on each area.

With a sterilized and cooled inoculating loop, small amount of the culture was picked
from the nutrient agar slant or Petri plate.
43

One or two colonies were emulsified on each drop to make a smooth suspension. The
smear should be about the size of a pea.

With a Pasteur pipette, one drop of hydrogen peroxide was placed over the test smear. Be
careful not to run the drops together.

Do not put anything in the other drop that serves as control.

The Fluid was observed over the smears for the appearance of gas bubbles.

The slide was discarded in a jar of disinfectant.

Limitations:
Hydrogen peroxide is unstable and should undergo a control check daily prior to use.

Growth for catalase testing must be taken from an 18-24 hour culture. Organisms lose
their catalase activity with age, resulting in a false-negative reaction.

Catalase activity is a function of aerobic process. Organisms incubated anaerobically

must be exposed to atmospheric oxygen for a minimum of 30 minutes before a catalase
test is performed. Failure to complete this step may produce false-negative results.

A positive catalase reaction with anaerobic organisms may be delayed for up to a minute
after addition of the reagent.

1. Coagulase Test:
Procedure: The enzyme coagulase is demonstrated in-vitro by two methods:
a) The Slide coagulase test
b) The Tube coagulase test

The Slide coagulase test:

Principle: This method measures bound coagulase. The bound coagulase is also known
as clumping factor. It cross-links the and chain of fibrinogen in plasma to form fibrin
clot that deposits on the cell wall. As a result, individual coccus stick to each other and
clumping is observed.

44

Procedure:
The slide was divided into two sections with grease pencil. One was labeled as test and
the other as control.

A small drop of distilled water was placed on each area.

One or two colonies of organism were emulsified on soya agar plate on each drop to
make a smooth suspension.

The test suspension was treated with a drop of citrated plasma and mixed well with a
needle.

Do not put anything in the other drop that serves as control. The control suspension
serves to rule out false positivity due to auto agglutination.

Clumping of organism within 5-10 seconds was taken as positive.

The Tube Coagulase Test:

Principle: This method helps to measure free coagulase. The free coagulase secreted by
S.aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which
is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.

Procedure:
Three test tubes were taken and labeled test, negative control and positive control.

Each tube was filled with 1 ml of 1 in 10 diluted rabbit plasma.

To the tube labeled test, 0.2 ml of overnight broth culture of test bacteria was added.

To the tube labeled positive control, 0.2 ml of overnight broth culture of known S.aureus
was added

To the tube labeled negative control, 0.2ml of sterile broth was added.

All the tubes were incubated at 37oC and observed the suspensions at half hourly
intervals for a period of four hours.

Positive result was indicated by gelling of the plasma, which remains in place even after
inverting the tube.

If the test remains negative until four hours at 37oC, the tube was kept at room
temperature for overnight incubation.

45

Limitations:

The slide test should be read very quickly, as false positives can occur.

Auto agglutination may occur.

Use water instead of saline for mixing.

The slide test should not be performed with organisms taken from high-salt media such as
Mannitol Salt Agar, as the salt content can create false positives.

Over mixing may cause the clot to break down.

The tube test is more reliable than the slide test.

We generally dont use the coagulase test when identifying unknowns.

Samples must be observed for clotting within 24 hours. This is because some strains that
produce coagulase also produce an enzyme called fibrinolysin, which can dissolve the
clot. Therefore, the absence of a clot after 24 hours is no guarantee that a clot never
formed. The formation of a clot by 12 hours and the subsequent disappearance of the clot
by 24 hours could produce a so-called false negative if the test were only observed at the
24-hour time.

3. Oxidase Test:
Procedure:
A filter paper soaked with the substrate tetramethyl-p-phenylenediaminedihydrochloride
was taken.

The paper was moistened with a sterile distilled water

The colony to be tested was picked with wooden or platinum loop and smear in the filter
paper

Inoculated area of paper was observed for a color change to deep blue or purple within
10-30 seconds

Precaution:
Do not use Nickel-base alloy wires containing chromium and iron (nichrome) to pick the
colony and make smear as this may give false positive results

Interpret the results within 10 seconds, timing is critical

46

IMViC TEST:

Indole test:

Reagents:

The main requirement for a suitable indole test medium is that it contain a sufficient
amount of tryptophan. Although many media meet this criterion, tryptone broth is
commonly used.
Tryptone broth

Ingredient

Amount

Tryptone

10.0 g

Sodium chloride

5.0 g

Dissolve the ingredients in 1 liter of sterile water. Dispense 4 ml per tube. Cap tube and
autoclave at 121oC under 15 psi pressure for 15 minutes. Store the tubes in the
refrigerator at 4 to 10C.

Kovcs reagent

Ingredient

Amount

Amyl or isoamyl alcohol, reagent grade

(butyl alcohol may be substituted)

150.0 ml

p-dimethylaminobenzaldehyde (DMAB)

10.0 g

HCl (concentrated)

50.0 ml

Dissolve DMAB in the alcohol. Gentle heating might be required to get the aldehyde
into solution.
47

Slowly add the acid to the aldehyde-alcohol mixture. The solution should be a pale
yellow color and is only stable for a short time. Store the mixture in a brown glass bottle
in the refrigerator. Kovcs reagent also is commercially available.

PROTOCOL
The tube of tryptone broth was inoculated with a small amount of a pure culture.

It was incubate at 35C (+/- 2C) for 24 to 48 hours.

To test for indole production, 5 drops of Kovcs reagent was added directly to the tube

A positive indole test was indicated by the formation of a pink to red color ("cherry-red
ring") in the reagent layer on top of the medium within seconds of adding the reagent.

If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy

Ehrlich's reagent

Ehrlich's reagent, an alternative to Kovcs reagent, also contains DMAB, which reacts
with indole to produce a red product. The Ehrlich formulation is more sensitive but
contains additional toxic or flammable solvents; it is recommended when testing bacterial
groups that produce little indole such as nonfermentative bacilli or anaerobes. Kovcs
reagent is apparently more stable and the absence of the additional organic extraction
(required with Ehrlich's) makes Kovcs formulation more suitable for undergraduate.

Ehrlichs reagent

Ingredients

Amount

Ethyl alcohol (absolute)

95.0 ml

p-dimethylaminobenzaldehyde
(DMAB)

HCl (concentrated)

1.0 g
20.0 ml
48

Dissolve components and store solution at room temperature in a brown glass bottle.

To test for indole production, inoculate a 4-ml tryptone broth tube with one loopful of
culture. After 24 to 48 hours of incubation, add to the culture 1.0 ml of either ether or
xylene. Mix well and then allow organic solvent to rise to the top of the medium. Add
0.5 ml of Ehrlich's reagent so that it runs down the side of the tube into the medium.
Development of a red color in the reagent layer indicates a positive test. If the reagent
stays yellow, the test is negative.

Indole spot test

Inoculate the bacterium to be tested on an agar medium that contains tryptophan.

Trypticase soy agar or sheep blood agar can be used. Incubate for 18 to 24 hours at the
appropriate temperature to allow for growth.

To conduct the test, place a small piece of Whatman filter paper in a petri dish cover.
Saturate paper with Kovcs reagent (1 to 1.5 ml). Smear the paper with cell paste from
an 18- to 24-hour culture.

If indole is present, a red pink color will develop within 1 to 3 minutes.

Methyl red test

Medium used
The medium used isMRVP broth,a nutrient medium with 0.5%glucoseadded. This
medium is also used for the Voges-Proskauer test, which determines whether neutral
fermentation products result from growth.

Procedure:
An inoculum from a pure culture was transferred aseptically to a sterile tube ofMRVP
broth.The inoculated tube was incubated at 35-37C for 24 hours.
49

Reagents added:

After incubation, five drops ofmethyl red were added. It is a pHindicator which is yellow
at neutral pHbut turns red at pH<4.0. Mixed acids fermentation results in a red color
change.

Inoculation of Medium

Select the MRVP broth medium.

Start your Bunsen burner.

Select the inoculating loop tool.

Flame your inoculating loop to sterilize it.

Remove the caps from your test tubes.

Flame the mouths of your test tubes.

Use the sterile inoculating tool to pick up an inoculum from the culture tube of the
unknown bacterium.

Immediately transfer the inoculum into the fresh, sterile medium.

Flame the mouths of your tubes once again.

Replace the caps on the test tubes.

Re-flame the inoculating tool.

Incubation of the Inoculated Medium

Place the inoculated tube into the 35-37 C incubator.

Incubate for the appropriate length of time. This test is properly interpreted after 24
hours.

Retrieve desired incubated culture from the incubator.

Addition of Reagents

Select the dropper tool and the appropriate reagent needed from the chemical shelf. For
this test, select methyl red.

Remove the cap from the tube.

50

Place the end of the dropper into the tube and add the reagent to the culture.

Determination of Test Results

Observe test result. For this test, addition of the appropriate reagent makes the culture
turn red if the test is positive. If negative, no color change is observed.

Record test result.

Dispose of the culture.

Voges-Proskauer Test

Culture:

24-48 hour tryptic soy broth culture.

Media:
MR-VP medium

Media preparation:
Weigh 5 g of glucose, 5 g of peptone and 5 g of dipotassium hydrogen phosphate
separately. Suspend all the ingredients in distilled water. Make up to 1000 ml. pH should
be 6.9. Dispense 3 ml of the media into each test tube, which are plugged and sterilized at
121C

Reagents:

Barritt's reagents A and B.

Preparation of Barritt's reagent

It consists of two solutions;

Solution A is prepared by dissolving 6 grams of a-naphtholin in 100 ml of 95% ethyl

alcohol.

Solution B is prepared by dissolving 16 grams of potassium hydroxide in 100 ml of

water.

51

Equipments:

Bunsen burner.
Inoculating loop

Procedure:
Using sterile technique, experimental organism was inoculated to the appropriately
labeled tube of medium by means of loop inoculation. The cultures were incubated for
24-48 hours at 37C. The experiment should be conducted in the LAF. Arrange the
materials required for the experiment in the LAF.

The loop was steilized vertically in the blue flame of the Bunsen burner till red hot. Heat
from the base of the wire was given first and slowly moved towards the loop (tip). The
wire was heated until it was red-hot.

From the rack, the test tube containing the Tryptic Soy Broth(TSB) cultures was taken
that has been kept for 24 - 48 hours.

The cap was removed from the TSB tube and the neck of the tube was flamed.

Using aseptic technique loop full of the organism was teken from the TSB (tryptic soy
broth).

Again the neck of the tube was flamed and the cap was replaced and the tube was placed
in the test tube rack.

Two sterile MR-VP broth tubes were taken, one named Test and the other Control.

Then we removed the cap of the MR-VP broth tube named 'Test' and flamed the neck of
the tube.

The MR-VP broth was inoculated with the inoculation loop containing the inoculum from
the TSB.

Again the neck of the MR-VP tube was flamed and placed in the test tube rack. Only the
broth in the tube named 'Test' was inoculatedusing aseptic technique. The broth in the
tube named 'Control' was left uninoculated.

Both the tubes (Test and Control) were incubated for 24 to 48 hours at 37C.

Broths were removed from the incubator.

The cap was removed and 10 drops of Barritt'sA reagent and 10 drops of Barritt's B
reagent were added to each broth.
52

It was shaked gently for several minutes.

Red color formation within 15 to 20 minutes is a positive result. No red color formation
after 15 to 20 minutes is a negative result.

Positive Result
Glucose ------Glucose Metabolism-------> Pyruvic Acid.
Pyruvic acid --------------->Acetoin.
Acetoin + added alpha-naphthol + added KOH = red color

Negative Result:
Glucose ------Glucose Metabolism-------> Pyruvic Acid.
Pyruvic acid -----------------> No Acetoin.
No acetoin + added alpha-naphthol + added KOH = copper color

Limitations:
Results of the MR and VP tests need to be used in conjunction with other biochemical
tests to differentiate genus and species within the Enterobacteriaceae.

A precipitate may form in the potassium hydroxide reagent solution. This precipitate has
not been shown to reduce the effectiveness of the reagent.

Most members of the family Enterobacteriaceae give either a positive MR test or a

positive VP test. However, certain organisms such as Hafniaalvei and Proteus mirabilis
may give a positive result for both tests.

Read the VP test at 48 hours. Increased incubation may produce acid conditions in the
broth that will interfere with the readings of the results.

VP reagents must be added in the order and the amounts specified or a weak-positive or
false-negative reaction may occur. A weak-positive reaction may be masked by a copperlike color which may form due to the reaction of KOH and a-naphthol.

Read the VP test within 1 hour of adding the reagents. The KOH and a-naphthol may
react to form a copper-like color, causing a potential false-positive interpretation.

Due to the possible presence of acetoin, diacetyl or related substances in certain raw
materials, the use of media low in these substances (such as MR-VP media) is
recommended for this test.

53

Citrate utilization test

Procedure of citrate utilization test:
Inoculate Simmons Citrate Agar lightly on the slant by touching the tip of a needle to a
colony that is 18 to 24 hours old.

Incubate at 35oC to 37oC for 18 to 24 hours. Some organisms may require up to 7 days of
incubation due to their limited rate of growth on citrate medium.

Observe the development of blue color; denoting alkalinization.

Expected Results:

Positive : Growth on the medium even without colour change will be considered as
positive.A colour change in the medium would be observed if the test organism produces
acid or alkali during its growth. The usual colour change observed is from green (neutral)
to blue (alkaline).

Negative : No growth observed.

Isolation of samples on SDA media( Sabouraud Dextrose Agar)

The samples were taken and were isolated differently on the two plates of SDA

These plates were kept for 2-3 days and the growth was observed

This was repeated for 2-3 times to get a proper growth

The anaerobic sample showed a lot of contamination

The aerobic sample was inoculated using streaking method to get a proper growth

54

Analysis of ETP:

Chemical Oxygen Demand (COD)

Introduction:
The industrial and municipal waste water effluents may contain very high amounts of organic
matter and if discharged into natural water bodies, it can cause complete depletion of dissolved
oxygen leading to the mortality of aquatic organisms. The amount of oxygen needed to consume
the organic and inorganic materials is called the Chemical Oxygen Demand (COD). Potassium
dichromate is considered the best oxidant due to its strong oxidizing ability, its applicability to a
wide variety of samples and ease of manipulation makes it very efficient.
Reagents used:
1. Potassium dichromate (Standard solution): K2Cr2O7 0.004167 M (0.0250 N)
2. Mohrs Salt: Ferrous ammonium sulphate (Standard solution): FeSO4.(NH4)2SO4(0.025 M)
3. Mercuric Sulphate: Powdered HgSO4
4. Silver Sulphate: Powdered Ag2SO4
5. Phenanthroline ferrous sulphate indicator solution
6. Concentrated Sulphuric acid: H2SO4 18 M
Procedure:
50 ml of sample was taken into a refluxing flask and several boiling stones were added. 0.1 g
HgSO4 was added to the solution. 5 ml of concentrated H2SO4 was also added to the solution.
To ensure that HgSO4 dissolved completely, the solution was swirled slowly while adding
Sulphuric acid. 0.1 g of Ag2SO4 was added to this solution. Finally Potassium dichromate was
added. Thorough mixing of the solution was ensured by swirling the flask in a water bath to
recover any volatile substances that may have escaped from the liquid state. The flask was then
attached to the condenser and further cooling was done. 20 ml of Sulphuric acid was added to the
solution in the flask continuing cooling and swirling to mix the solution. The solution was
refluxed for 1 hour.

55

A blank run (using 50 ml distilled water instead of sample) was simultaneously conducted with
the same procedure after cooling; the solution was transferred to an Erlenmeyer flask. The reflux
flask was rinsed thrice, pouring the rinsing water to the Erlenmeyer flask. The solution was
diluted to about 300 ml and about 8 drops of Phenanthroline ferrous sulphate was added to the
solution as an indicator. The solution was titrated against the Mohrs salt and the titer volume
required for the color change from blue-green to reddish blue was noted.The procedure was
repeated for the blank run.
Observations:
Solution
Sample
Blank

Intial reading (ml)

0.00
0.00

Final reading(ml)
19.60
21.20

Titar volume(ml)
Vs
Vb

Calculations:
COD = 8000 * (Vb Vs)* M/ original volume of sample taken mg/l
Where,
Vb = Titer volume for the blank
Vs = Titer volume for the sample
M = Molarity of Mohrs solution
COD = 8000 * (21.20-19.60) * 0.025/ 50
= 6.4 mg/l

Discussions and ResultsPotassium dichromate acts as a strong oxidizing agent and oxidizes the organic and inorganic
matter in the waste water. The reaction can be expressed as Cr2O72 - + 14 H+ + 5 e- = 2 Cr+ + 7 H2O
If chlorides are present in the sample it will interfere with the oxidation of the organic matter. To
ensure non-interference of chlorides Mercury Sulphate is added which will form complex of
mercuric chloride. An amount of 10 g of Mercury Sulphate is required for 1 g of Chlorides to
form complex.

56

Sulphuric acid is added to the mixture so that the mercury is completely dissolved. Besides, it
assists in oxidizing the nitrogen compounds in the sample and the increased heat will accelerate
the reaction rate.
Silver Sulphate catalyses the reaction and also assists in the oxidation of the nitrogen
compounds.
Mercury sulphate is added first in order to allow the chlorine atoms to combine with mercury. If
Silver Sulphate is added first, the chlorine would bind with the silver. Mercury sulphate may be
added after; however it will take some time for the chlorine to detach from the silver and bind to
mercury. Thus, it is best to add mercury sulphate first.
The titer volume of the sample gives the volume of Ferrous Ammonium Sulphate required to
react with the excess potassium dichromate in the solution. Similarly, the titer volume for the
blank (distilled water) gives the volume of Ferrous Ammonium Sulphate required to react with
the excess potassium dichromate in the blank. The equation for the titration can be expressed as:
Cr2O72 + FeSO4 (NH4)2SO4 = Cr+ + NH4+ + Fe 3+
From above equation it can be seen that one molecule of dichromate corresponds to one
molecule of Mohrs salt. Thus, the difference in volume of excess K2Cr2O7 reacting with
Mohrs solution can be calculated from the expression:
= (Original vol. K2Cr2O7 vol. of K2Cr2O7 used for oxidation) solution - (Original vol.
K2Cr2O7 vol. of K2Cr2O7 used for oxidation) blank
= (Vol. of K2Cr2O7 used for oxidation) blank (Vol. of K2Cr2O7 used for oxidation) solution
Hence, the difference in the titer volume for the solution and the blank is used to find out the
Chemical Oxygen Demand directly.
Conclusion
COD of given sample using the method of titration is found to be 6.4 mg/l.

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Biological Oxygen Demand:

Principle: The method consists of filling with sample, to overflowing, an airtight bottle of the
specified size and incubating it at the specified temperature for 5 d. Dissolved oxygen
ismeasured initially and after incubation, and the BOD is computed from the difference between
initial and final DO. Because the initial DO is determined shortly after the dilution is made, all
oxygen uptake occurring after this measurement is included in the BOD measurement.
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
gNa2HPO47H2O, 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 1L.
e. Acid and alkali solutions, 1N, for neutralization of caustic or acidic waste samples.
1) AcidSlowly and while stirring, add 28 mL conc sulfuric acid to distilled water. Dilute
to 1 L.
2) AlkaliDissolve 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.

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h. Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade glutamic acid at
103C 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 sampledilutions.
Procedure:
a.
b.
c.
d.
e.
f.
g.
h.
i.

Preparation of dilution water

Dilution water storage
Glucose-glutamic acid check
Seeding: Seed source, Seed control
Sample pre-treatment
Dilution techniques
Determination of initial DO
Incubation
Determination of final DO

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

When dilution water is seeded:

where:
D1 = DO of diluted sample immediately after preparation, mg/L,
D2 = DO of diluted sample after 5 d incubation at 20C, mg/L,
P = decimal volumetric fraction of sample used,
B1 = DO of seed control before incubation, mg/L

59

B2 = DO of seed control after incubation mg/L and

f = ratio of seed in diluted sample to seed in seed control = (% seed in diluted sample)/(% seed in
seed control). If seed material is added directly to sample or to seed control bottles:
f = (volume of seed in diluted sample)/(volume of seed in seed control)
report results as CBOD if nitrification is inhibited.
If more than one sample dilution meets the criteria of a residual DO of at least 1 mg/L and a DO
depletion of at least 2 mg/L and there is no evidence of toxicity at higher sample concentrations
or the existence of an obvious anomaly, average results in the acceptable range. In these
calculations, do not make corrections for DO uptake by the dilution water blank during
incubation. This correction is unnecessary if dilution water meets the blank criteria stipulated
above. If the dilution water does not meet these criteria, proper corrections are difficult ; do not
record results or, as a minimum, mark them as not meeting quality control criteria.

Total Suspended Solids (TSS)

Total suspended solids (TSS) include all particles suspended in water which will not pass
through a filter. Suspended solids are present in sanitary wastewater and many types of industrial
wastewater. There are also nonpoint sources of suspended solids, such as soil erosion from
agricultural and construction sites. As levels of TSS increase, a water body begins to lose its
ability to support a diversity of aquatic life. Suspended solids absorb heat from sunlight, which
increases water temperature and subsequently decreases levels of dissolved oxygen (warmer
water holds less oxygen than cooler water). Some cold water species, such as trout and
stoneflies, are especially sensitive to changes in dissolved oxygen. Photosynthesis also decreases,
since less light penetrates the water. As less oxygen is produced by plants and algae, there is a
further drop in dissolved oxygen levels. TSS can also destroy fish habitat because suspended
solids settle to the bottom and can eventually blanket the river bed. Suspended solids can
smother the eggs of fish and aquatic insects, and can suffocate newly-hatched insect larvae.
Suspended solids can also harm fish directly by clogging gills, reducing growth rates, and
lowering resistance to disease. Changes to the aquatic environment may result in a diminished

60

food sources, and increased difficulties in finding food. Natural movements and migrations of
aquatic populations may be disrupted.
Procedure:
Take a filter paper, dry it in oven at 1050 C temp. for 1 hr. weigh it (intial weight) now take 50ml
of well shaked samples, if suspended solids are of low range, take 100 ml of sample or so and
filter the sample. Dry the filter paper along with resiue in oven at 1050 C cool it and weigh it to a
constant weight (Final Weight).
Calculations;
T.S.S in ppm = W/V x 106

Where,
W = Final weight of filter paper (with solids ) - intial weight of filter paper (without solids)
V = Volume of samples used

For AERATION TANK : TSS: ((1.117-1.044)/50)*10^6

i.e 1460 mg/l.
For SAFF TANK : TSS: ((1.027-1.019)/50)*10^6
i.e 160 mg/l.

Total Dissolved Solids (TDS)

Solids are found in streams in two forms, suspended and dissolved. Suspended solids include silt,
stirred-up bottom sediment, decaying plant matter, or sewage-treatment effluent. Suspended
solids will not pass through a filter, whereas dissolved solids will. Dissolved solids in freshwater
samples include soluble salts that yield ions such as sodium (Na+), calcium (Ca2+), magnesium
(Mg2+), bicarbonate (HCO3), sulfate (SO42 ), or chloride (Cl). Total dissolved solids, or
TDS, can be determined by evaporating a pre-filtered sample to dryness, and then finding the
mass of the dry residue per liter of sample. A second method uses a Vernier Conductivity Probe
to determine the ability of the dissolved salts and their resulting ions in an unfiltered sample to
conduct an electrical current. The conductivity is then converted to TDS. Either of these methods
yields a TDS value in units of mg/L.
Sources of Total Dissolved Solids
Hard-Water Ions
61

- Ca2+, Mg2+, HCO3

Fertilizer in agricultural runoff
- NH4+, NO3, PO43, SO42
Urban runoff
- Na+, Cl
Salinity from tidal mixing, minerals, or returned irrigation water
- Na+, K+, Cl
Acidic rainfall
- H+, NO3, SO32, SO42
TDS is sometimes used as a watchdog environmental test. Any change in the ionic
composition between testing sites in a stream can quickly be detected using a Conductivity
Probe. TDS values will change when ions are introduced to water from salts, acids, bases, hardwater minerals, or soluble gases that ionize in solution.
If TDS levels are high, especially due to dissolved salts, many forms of aquatic life are affected.
The salts act to dehydrate the skin of animals. High concentrations of dissolved solids can add a
laxative effect to water or cause the water to have an unpleasant mineral taste. It is also possible
for dissolved ions to affect the pH of a body of water, which in turn may influence the health of
aquatic species. If high TDS readings are due to hard-water ions, then soaps may be less
effective, or significant boiler plating may occur in heating pipes.
Summary of Methods
Method 1: TDS Using a Conductivity Probe
A Vernier Conductivity Probe is used on site, or placed into samples collected at sites, to
measure TDS concentration of the solution. It offers the advantage that it can be performed
without filtration, providing instantaneous feedback about total dissolved solids concentration in
a stream.
Method 2: TDS by Evaporation
Using this method, samples are first filtered to remove suspended solids. A precise amount of
sample is added to a carefully cleaned, dried, and weighed beaker. The water is then evaporated
in a drying oven at 1050 C. The difference in mass between the two weighings is the mass of the
62

total dissolved solids. Calculations are then performed to convert the change in mass to mg/L of
TDS. This procedure does not require a sensor, but does require an analytical balance (0.001 or
0.0001-g resolution).

Results and Discussions:

Biochemical tests:

For first pure culture of aerobic sample i.e AP 1

Catalase test:

Slant method:
There was a formation of bubbles and foam in the slant.

Slide method:
There was a formation of bubbles on slide.

Coagulase test:

Tube method:
Gelling of plasma took place which resulted in immobilized clump even after inverting it.

63

Slide method:
Coagulation of the microorganism takes place.

Oxidase test:

The color doesnt change to deep blue purple. Result is oxidase negative.

IMViC test:
1. Indole test:
There is no change in color in the layer of reagent. So it is indole negative.

2. Methyl red test:

A color changes to red when methyl red indicator is added to reagent

3. Voges Proskauer test:

A maroon band is found after addition of Barits reagent A and B

4. Citrate utilization test

The color remains dark forest green. So it is citrate utilization test negative

64

2. For second pure culture of aerobic sample i.e AP2

Catalase test:

Slant method:
There was a formation of bubbles and foam in the slant.

Slide method:
There was a formation of bubbles on slide.

Coagulase test:
No clumps formation takes place in any of the tube test and slide test
So it is coagulae negative

Oxidase test:
Change of color take place to deep blue purple.

65

IMViC Test:
1. Indole test:
There is no change in color in the layer of reagent. So it is indole negative.

2. Methyl red test:

There is no formation of red color after adding methyl red indicator.

3. Voges Proskauer test:

There is no formation of maroon band after addition of Baritts reagent

4. Citrate utilization test:

The color changes to blue from dark forest green. It is citrate positive.

3. For third pure culture of aerobic sample i.e AP3

Catalase test:
Slant test:
There is formation of bubbles in slant.

Slide test:
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 On slide there was formation of bubbles after addition of H2O2.

Coagulase test:
There is no formation of clumps in any of the methods i.e tube and slide method.

Oxidase test:
There is no formation of deep blue color so it is oxidase negative

IMViC test:
1. Indole test:
It shows positive result for indole test. A pink to red color forms in the reagent layer.

2. Methyl red test:

It changes the color after addition of methyl red indicator

67

3. Voges Proskauer test:

There is no formation of maroon band after addition of Baritts reagent A and B

4. Citrate utilization test:

The color doesnt changes to blue. It remains dark forest green.

4. For first pure culture of anaerobic sample i.e ANP1

Catalase test:
Slant method:
It is a catalase positive organism due to formation of bubbles

68

Slide method:
Bubble formation takes place

Coagulase test:
There are no clumps formed in this process. So it is a coagulase negative microorganism

Oxidase test:
There was no dark blue purple formation. So it is oxidase negative organism

IMViC test
1. Indole test:
There is no change in color in the layer of reagent. So it is indole negative.

2. Methyl red test:

There is no formation of red color after addition of methyl red indicator.

69

3. Voges Proskauer test:

A maroon band is found after addition of Barits reagent A and B

4. Citrate utilization test:

The color changes to blue from dark forest green. It is citrate positive.

5. For second pure culture of anaerobic sample i.e ANP2

Catalase test:
Slant test:
There is formation of bubbles in slant.

Slide test:
On slide there was formation of bubbles after addition of H2O2.

Coagulase test:
70

 There is no formation of clumps in any of the methods i.e tube and slide method.

Oxidase test:
There is no formation of deep blue color so it is oxidase negative

IMViC test:
1. Indole test:
It shows positive result for indole test. A pink to red color forms in the reagent layer.

2. Methyl red test:

It changes the color after addition of methyl red indicator

3. Voges Proskauer test:

There is no formation of maroon band after addition of Baritts reagent A and B

71

4. Citrate utilization test:

The color doesnt changes to blue. It remains dark forest green.

Miroorganisms Detected:
1. Microorganism from First pure culture of aerobic sample i.e AP 1 was detected as
Staphylococcus aureus based on the morphology and the biochemical tests.
2. Microorganism from second pure culture of aerobic sample i.e AP2 was detected
as Pseudomonas aeruginosa based on the morphology and biochemical tests.
3. Microorganism from Third pure culture of aerobic sample i.e AP3 was detected as
Escherichia coli based on the morphology and biochemical tests.
4. Microorganism from First pure culture of anaerobic sample i.e ANP1 was
detected as Klebsiella pneumonia based on the basis of morphology and
biochemical tests.
5. Microorganism from Second pure culture of anaerobic sample i.e ANP2 was
detected as Escherichia coli based on the morphology and biochemical tests.

72

 The aerobic sample was plated on SDA as well with help of streaking method.
It was incubated for some days.
There was formation of fungus i.e Aspergillus niger in the plate.

Analysis of ETP:
Values
BOD:
For Treated water: 156 mg/l
For Anaerobic water: 1475 mg/l
COD: Value of COD of sampling tank: 6.4 mg/l
TSS:

TDS:

Value of sample of Aeration tank came out to be 1460 mg/l

Value of sample of SAFF tank came out to be 160mg/l
Value of sample of Treated water: 2770 mg/l
Value of sample of Anaerobic water: 3250mg/l

73

References

Dennis R. Heldman, Encyclopedia of Agricultural, Food, and Biological Engineering

(2003), p. 55.

Khopkar, S. M. (2004). Environmental Pollution Monitoring And Control. New Delhi:

New Age International. p. 299. ISBN 81-224-1507-5.

http://www.slideshare.net/kps_senthil/biochemical-test-of-bacteria.

http://en.wikipedia.org/wiki/IMViC

http://microbeonline.com/imvic-tests-principle-procedure-and-results/

http://www.slideshare.net/JaidevSingh/effluent-treatment-plant-design-operation-andanalysis-of-waste-water-16567872

http://www.microbelibrary.org/library/laboratory-test/3006-biochemical-test-media-forlab-unknown-identification

http://onlinelibrary.wiley.com/doi/10.1111/j.1348-0421.1970.tb00490.x/abstract

http://en.wikipedia.org/wiki/Wastewater_treatment_plant

http://www.biotechservices.in/waste-water-treatement-plants.html

http://www.cpet.ufl.edu/wp-content/uploads/2013/03/Identifying-Bacterial-Unknownsusing-Biochemical-Tests.pdf

http://ebookbrowsee.net/identification-of-bacteria-by-biochemical-testing-docd190114641

http://www.ais.unwater.org/ais/pluginfile.php/356/mod_page/content/111/CountryReport
_India.pdf

http://www.indiaenvironmentportal.org.in/category/1206/thesaurus/effluent-disposalstandards/

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