Professional Documents
Culture Documents
B.Tech Biotechnology
(July-December, 2013)
Submitted by
Sahib Bhanot
Roll No: 701000033
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
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
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.
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
Physical
Primary
Secondary
Biological and
chemical
11
Tertiary/advanced
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
Taste and odor control:-Activated carbon, chlorine, chlorine dioxide, copper sulphate,
ozone, potassium permanganate.
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
30
350
100
Boron
Cadmium
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
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
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).
Catalase test
Coagulase test
Oxidase test
IMViC test
1. Indole test
2. Methyl red test
3. Voges-Proskauer test
4. Citrate utilization test
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.
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
lactose
fermenters.
Salmonella
typhimurium,
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.
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.
The floor, workbench, and instruments shall be mopped with dry cloth
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
1.5
Phenyl
2.5
The disinfectants prepared shall be used for one day only and shall be
discarded after use
Entry and Exit in Microbiology lab should be documented with time and date
Dip the hands and feet in disinfectant solution kept in change room 1
Take the set of sterile garments kept in garment cubicle without allowing them
to come in contact wih floor or walls
Nose mask
Head cover
Shoe cover
Dont touch the center surface of the gloves with finger and palm
24
Add detergents like Extran which is suface active and should be used as per
label instructions
Scrub all the glassware by brush in such a way that all remaining from inner
surface are washed out
Place the glassware in inverted position on SS trays to drain out excess water
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
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
Collection of samples: The samples from effluent treatment plant were collected from
both anaerobic and aerobic tank for the identification.
36
Gms / Litre
15.000
5.000
5.000
15.000
7.30.2
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
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
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).
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
Catalase Test:
Material Required:
Cultures: 24-48 hour tryptic soy broth cultures of bacteria
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.
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.
With a sterilized and cooled inoculating loop, small amount of the culture was picked
from the nutrient agar slant or Petri plate.
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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.
The Fluid was observed over the smears for the appearance of gas bubbles.
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.
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
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Procedure:
The slide was divided into two sections with grease pencil. One was labeled as test and
the other as control.
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.
Procedure:
Three test tubes were taken and labeled test, negative control and positive control.
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.
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Limitations:
The slide test should be read very quickly, as false positives can occur.
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.
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 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
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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
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.
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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.
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
95.0 ml
p-dimethylaminobenzaldehyde
(DMAB)
HCl (concentrated)
1.0 g
20.0 ml
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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.
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.
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.
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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
Use the sterile inoculating tool to pick up an inoculum from the culture tube of the
unknown bacterium.
Incubate for the appropriate length of time. This test is properly interpreted after 24
hours.
Addition of Reagents
Select the dropper tool and the appropriate reagent needed from the chemical shelf. For
this test, select methyl red.
Place the end of the dropper into the tube and add the reagent to the culture.
Voges-Proskauer Test
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:
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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.
The cap was removed and 10 drops of Barritt'sA reagent and 10 drops of Barritt's B
reagent were added to each broth.
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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.
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.
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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.
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).
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
The aerobic sample was inoculated using streaking method to get a proper growth
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Analysis of ETP:
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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
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.
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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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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.
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:
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
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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
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).
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.
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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.
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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.
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IMViC Test:
1. Indole test:
There is no change in color in the layer of reagent. So it is indole negative.
Slide test:
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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.
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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.
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Slide test:
On slide there was formation of bubbles after addition of H2O2.
Coagulase test:
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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.
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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.
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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:
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References
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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