Contents

1. Introduction
2. Review of literature
3. Materials and methods
4. Results and analysis
5. Conclusion
6. Reference
INTRODUCTION
Water is one of the most important of all natural resources known on earth. It is
important to all living organisms, most ecological systems, human health, food
production and economic development (Postel et al., 1996). The safety of drinking
water is an on going concern within the global village. Traditionally, the safety of
potable water supplies has been controlled by disinfection, usually by chlorination
and coliform population estimates. However, it has been reported that coliform-
free potable water may not necessarily be free of pathogens (Sim et al., 1987).
Many congenital diseases such as goiter and cancer have been associated with
presence of high concentration of a chemical or its inadequate supply in water.
Opinya et al, (1987) reported that low or high level of fluoride ions concentration
in water as the major cause of dental flurosis. Low concentration of iodine in
Homo sapiens results in goiter. Infants have been considered as a potential high
risk group to the toxic effects of sodium from drinking water (smith, 1974).
Currently, about 20% of the world’s population lacks access to safe drinking water,
and more than 5 million people die annually from illness associated with safe
drinking water or inadequate sanitation. If everyone had safe drinking water and
adequate sanitation services, there would be 200milion fewer cases of diarrhea and
2.1 million fewer deaths caused by diarrhoeal illness each year (Hunter et al.,
2001). Biofilms in drinking water distribution system has generated health
concerns. Biofilms are coating of organic and inorganic materials in pipes that can
harbor, protect and allow the proliferation of several bacterial pathogens, including
Legionella and Mycobacterium avium complex (MAC). Factors that affect
bacterial growth on biofilms include water temperature, type of disinfectant and
residual concentration, biodegradable organic carbon level, degree of pipe
corrosion and treatment/distribution system characteristics. Chloramines are
considerably more effective than chlorine for controlling legionella in biofilms
distribution system deficiencies linked to a number of water born disease
outbreaks. (Hunter et al., 2001). Amazingly, current drinking water standards don’t
even require testing for any of the more than 7,000 pharmaceutical compounds
being prescribed; pharmaceutically active compounds such as analgesics,
antibiotics, antileptics, anti-rheumatics, beta blockers, chemotherapeutics, steroid
hormones and X-ray contrast media have been detected in tap water in Europe and
Americas (Bob Masters, 2001). The aim of this study was to evaluate the sanctity
of potable water in circulation within Lagos State University, Ojo Campus and
suggest safety measures to reduce the incidence of water – borne disease.

Water is one of the prime elements responsible for life on earth as two thirds of
earth’s surface is covered by water. Ninety seven percent of the world’s water is
found in Oceans. Only 2.5% of the world’s water is non-saline fresh water .
However, 75% of all the fresh water is bound up in glaciers and ice caps. Of the
remaining 25% fresh water is found in lakes, rivers and 24% is present as ground
water. Water is the essential resource for living system, industrial process,
agricultural production and domestic use. The use of water increases with growing
population, putting increasing strain on these water resources. An adequate supply
of safe drinking water is one of the major pre requisites for a healthy life. Pollution
occurs when a product added to our natural environment adversely affects nature’s
ability to dispose it off. A pollutant is something which adversely interferes with
health, comfort, property or environment of the people. Generally, most pollutants
are introduced in the environment as sewage, agricultural waste, domestic waste,
industrial waste, accidental discharge and as compounds used to protect plants and
animals. As a result of the increasing demand for water and shortage of supply, it is
necessary to increase the rate of water development in the world and to ensure that
the water is used more efficiently. Drinking water should be suitable for human
consumption and for all usual domestic purposes. The importance of water in daily
living makes it imperative that through examinations be conducted on it before
consumption . The determination of drinking water quality guideline value is
essential in order to avoid health risks to the consumers. In developing countries
only a small proportion of the waste water produced by severed communities is
treated. Developing country governments and their regulatory agencies, as well as
local authorities (which maybe city or town councils or specific waste water
treatment authorities or more generally waste and sewage authorities) need to
understand that domestic and other waste waters require treatment before discharge
or preferably recycle and reuse in agriculture or aquaculture. The qualities of water
need to be evaluated thoroughly to generate base line information for welfare of
society. It is necessary to isolate and identify the microorganisms present in the
different water samples. In order to alleviate microbial water pollution first a
systematic study on the types and concentration of microbes present in different
sources at different seasons is to be made. With this objective in view, the present
work is planned to assess the quality of water in and around Vijayawada city from
seven different sites for microbiological parameters and the results are compared
with the standards given by WHO, determined the extent of microbial pollution
and recommendations suggested to improve the quality of Krishna river water.

The global demand for quality water, whether for purposes of drinking, sanitation,
irrigation, and industrial use, has been on a continuous rise, and there has been
overwhelming concern in recent years about water treatment and reuse requiring
the strictest standards .The pharmaceutical industry is beset with high-value, low
volume multiproduct plants on one hand which are mostly batch operations
wherein the effluent is mixed and treated. There are some dedicated batch,
semibatch, and continuous process plants producing bulk drugs. These plants use
different types of reactants, (homogeneous) catalysts, solvents, solids, and water,
handled in special equipment. In these types of units, the major cost of the drug
depends on the type of impurity rather than on the purity of the drug. Thus,
separation processes play a very vital role in this industry. The so-called
environmental quotient or E-factor for the pharmaceutical industry is anywhere
between 50 and 100 kg/(kg of desired product) since these processes are multistep
operations (anywhere between 5 and 30 steps) with several noncatalytic routes
using copious quantities of (volatile organic compound (VOC)) solvents or “crazy”
mixtures of close boiling solvents. Further, ultrapure water is used in the
pharmaceutical sector to give multiple washings to the solid cake or to use as
extractant or as solvent per se. This water is not reused due to strict regulations as
defined in drug master file (DMF) etiquettes approved by the authorities. The
presence, outcome, and toxicity of pharmaceutical residues in the aquatic
environment pose difficulties. Therefore, recovery of high-value API and
pharmaceutical drugs from dilute streams, instead of treatment,ought to be
considered while dealing with this issue. Many of the frequently used generic
drugs such as antibiotics, analgesics, antihistamines, and antituberculosis (anti-TB)
drugs, etc., are used on the same scale as pesticides and other organic micro
pollutants, but they are not subjected to the same level of scrutiny for possible
environmental effects. The total spread and repercussions of the presence of these
moieties in the environment are therefore mostly unknown and ill-defined.
Although these compounds have been detected in a wide variety of environmental
samples including sewage, surface waters, groundwater, and potable water, their
concentrations generally range from a few parts per trillion to parts per billion
levels. It is therefore very often considered unlikely that pharmaceuticals will have
a detrimental effect on the environment. However, in the absence of validated
analytical methods, proper monitoring information, and associated data about the
fate and toxicity of the pharmaceutical compounds and/or their metabolites in the
aquatic environment, it is difficult to make a correct risk assessment. The purpose
of the current review is to take stock of effluent arising from different sectors of
active pharmaceutical ingredients (API), bulk drugs, and related pharmaceutics,
which use large quantities of water, to propose strategies to recover to a large
extent the valuable compounds, to demonstrate the economic benefit of recovery,
and finally to discuss the treatment of very dilute but detrimental wastewaters.
Some important drug manufacture flow sheets are included to show how and why
the waste is generated and whether some steps could be combined to reduce the
cost. There are instances where adequate data are not available or the industry
would not share such information for being targeted by pollution control
authorities. We also believe that there is tremendous scope to develop new
strategies for some of the old problems from the perspective of green chemistry
and waste minimization principles.

Water treatment describes those industrial-scale processes used to make water

more acceptable for a desired end-use. These can be use for drinking water,
industry, medical and many other uses. The goal of water treatment process is to
remove existing contaminants in the water. The processes involved in treating
water for drinking purpose may be solids separation using physical processes such
as settling and filtration, and chemical processes such as disinfection and
coagulation. Biological processes employed in the treatment of waste water and
these processes may include, for example, aerated lagoons, activated sludge or
sand filters.
SOURCES OF WATER

Ground water: The water emerges from some deep ground water may have fallen
as rain many hundreds, or thousands of years ago. Rock and soil and layers
naturally they filter the ground water to a high degree of clarity and often it does
not require additional treatment other than adding chlorine or chloramines as
secondary disinfectants. Such water may emerge as springs, artesian springs, or
may be extracted from boreholes or wells. Deep ground water is generally of very
high bacteriological quality (I.e., pathogenic bacteria or the pathogenic protozoa
are typically absent), but the water may be rich in dissolved solids, especially
carbonates and sulfates of calcium and magnesium. Depending on the strata
through which the water has flowed, other ions may also be present including
chloride, and bicarbonate. There may be a requirement to reduce the iron or
manganese content of this water to make it acceptable for drinking, cooking, and
laundry use.

Rivers, canals and low land reservoirs: Low land surface waters will have a
significant bacterial load and may also contain algae, suspended solids and a
variety of dissolved constituents. Atmospheric water generation is a new
technology that can provide high quality drinking water by extracting water from
the air by cooling the air and thus condensing water vapor. Rainwater harvesting or
fog collection which collects water from the atmosphere can be used especially in
areas with significant dry seasons and in areas which experience fog even when
there is little rain.
Desalination of seawater by distillation or reverse osmosis:

Surface Water: Freshwater bodies that are open to the atmosphere and are not
designated as groundwater are classified in the USA for regulatory and water
purification purposes as surface water.

Potable water treatment

Water purification is the removal of contaminants from untreated water to produce

drinking water that is pure enough for the most critical of its intended uses, usually
for human consumption. Substances that are removed during the process of
drinking water treatment include suspended solids, bacteria, algae, viruses, fungi,
minerals such as iron, manganese and sulphur, and other chemical pollutants such
as fertilisers.

Drinking water treatment

A combination selected from the following processes is used for municipal

drinking water treatment worldwide: Pre-chlorination - for algae control and
arresting any biological growth Aeration - along with pre-chlorination for removal
of dissolved iron and manganese Coagulation - for flocculation Coagulant aids,
also known as polyelectrolytes - to improve coagulation and for thicker floc
formation Sedimentation - for solids separation, that is, removal of suspended
solids trapped in the floc Filtration - removing particles from water Desalination -
Process of removing salt from the water Disinfection - for killing bacteria.
Sewage treatment

Sewage treatment is the process that removes the majority of the contaminants
from wastewater or sewage and produces both a liquid effluent suitable for
disposal to the natural environment and sludge. At the simplest level, treatment of
sewage and most wastewaters is carried out through separation of solids from
liquids, usually by sedimentation.

Industrial water treatment

Two of the main processes of industrial water treatment are boiler water treatment
and cooling water treatment. A lack of proper water treatment can lead to the
reaction of solids and bacteria within pipe work and boiler housing. Steam boilers
can suffer from scale or corrosion when left untreated leading to weak and
dangerous machinery, scale deposits can mean additional fuel is required to heat
the same level of water because of the drop in efficiency. Poor quality dirty water
can become a breeding ground for bacteria such as Legionella causing a risk to
public health. With the proper treatment, a significant proportion of industrial
onsite waste water might be reusable. This can save money in three ways: lower
charges for lower water consumption, lower charges for the smaller volume of
effluent water discharged and lower energy costs due to the recovery of heat in
recycled wastewater. Corrosion in low pressure boilers can be caused by dissolved
oxygen, acidity and excessive alkalinity. Water treatment therefore should remove
the dissolved oxygen and maintain the boiler water with the appropriate pH and
alkalinity levels. Without effective water treatment, a cooling water system can
suffer from scale formation, corrosion and fouling and may become a breeding
ground for harmful bacteria such as those that cause Legionnaires ‘disease. This
reduces efficiency, shortens plant life and makes operations unreliable and unsafe.
MATERIAL AND METHOD

MATERIALS:

Sterilized Bottle, 70% IPA , petri plate ,selective media(CA , MSA ,RVSB
,MCB ,MCA , XLDA , M-Endo) ,filter paper , inoculation loop ,Tissue paper,
Cotton, purified water etc.

Types of Nutrient Media Used In Analysis

Mac Conkey agar is culture medium designed to grow Gram-negative bacteria and
stain them for lactose fermentation. It contains bile salts (to inhibit most Gram-
positive bacteria), crystal violet dye (which also inhibits certain Gram-positive
bacteria), neutral red dye (which stains microbes fermenting
lactose), lactose and peptone. Alfred Theodore MacConkey developed it while
working as a bacteriologist for the Royal Commission on Sewage Disposal in
the United Kingdom.

Endo agar contains peptone, lactose, dipotassium phosphate, agar, sodium sulfite,

basic fuchsin and was originally developed for the isolation of Salmonella typhi,
but is now commonly used in water analysis. As in MacConkey agar, coliform
organisms ferment the lactose, and the colonies become red. Non-lactose-
fermenting organisms produce clear, colourless colonies against the faint pink
background of the medium.

mFC medium is used in membrane filtration and contains selective and differential
agents. These include rosolic acid to inhibit bacterial growth in general, except for
faecal coliforms, bile salts inhibit non-enteric bacteria and aniline blue indicates
the ability of faecal coliforms to ferment lactose to acid that causes a pH change in
the medium.[8]
TYEA medium contains tryptone, yeast extract, common salt and L-arabinose per
liter of glass distilled water and is a non selective medium usually cultivated at two
temperatures (22 and 36 °C) to determine a general level of contamination (a.k.a.
colony count).

INSTRUMENT

Microscope, Colony counter, Autoclave, Laminar air flow system,

Incubator, Drying Oven, Hot Air Oven for Sterilization, PH Meter,
Analytical Balance.

MICROSCOPE

It is a technical field using microscope as a tool for viewing sub-microscope

entities ,objects ,structures for better magnification which could not be
achieved by naked (unaided) eyes.
COLONY COUNTER

Colony counter are used to estimate a liquid culture’s density of microorganism by

counting individual colonies on an agar plate , slide ,or petri dish. The counting can
be accomplished manually, often with touch pressure and a digital counter, or can
be semi- or fully automatic.
AUTOCLAVE

Autoclave is the nucleus of a microbiology laboratory. It is used not only to

sterilize liquid substances such as prepared media and saline (diluents) solutions,
but also to sterilize glassware when required.

It has the same working principal as a domestic pressure cooker. The maximum
temperature that can be obtained by boiling water in an open container is 100°C
(boiling point of water).
LAMINAR AIR FIOW

Laminar air Flow is an enclosed bench designed to prevent contamination like

biological particles or any particles sensitive device.

This closed cabinet is usually made up of stainless steel without any gap or joints
where spores might collect.

Laminar hoods are equipped with a shortwave ultra violet germicidal lamp to
sterilize the shell.

USE

Laminar Air Flow provides a work area with aseptic/sterile conditions for the
tissue culture.
INCUBATOR

An incubator is a device used to grow and maintain microbiological cultures or cell

culture. The incubator maintains optimal temperature, humidity and other
conditions such as the co2 and oxygen content of the atmosphere inside. Incubators
are essential for a lot experimental work in cell biology, microbiology and
molecular biology and are used to culture both bacterial
HOT AIR OVEN

A hot air oven is a type of dry heat sterilization. Dry heat sterilization is used on
equipment that cannot be wet and on material that will not melt, catch fire, or
change form when exposed to high temperature. Moist heat sterilization uses water
to boil items or steam them to sterilize and does not take as long as dry heat
sterilization. Examples of items that are not sterilized in a hot air oven are surgical
dressings, rubber items , or plastic material.
HOT PLATE

In laboratory setting, hot plates are generally used to heat glassware or its
contents . Some hot plates also contain a magnetic stirrer , allowing the heated
liquid to be stirred automatically.
PH Meter

A pH meter is an electronic instrument used to measure the pH (acidity or basicity)

of a liquid (thought special probes are sometimes used to measure the pH of semi-
solid substances). A typical pH meter consists of a special measuring probe (a
glass electrode) connected to an electronic meter that measures and displays the pH
reading.
Analytical Balance

An analytical balance is the class of balance design to measure small mass in the
sub milligram range. The measuring pan of an analytical balance inside a
transparent enclosure with door so that dust dose not collect and so any currents in
the room do not affected the balance operation.
METHOD:-

PREPRATION OF SAMPLING BOTTLES

1. SAMPLING OF PURIFIDE WATRE FOR MICRO TESTING

2. MICROBIAL TESTING PARAMETERS FOR PURIFIDE WATER
 Total aerobic plate count (pour plate method)
 Total coliform count (membrane filtration)
 Test for specified microorganism
 Escherichia coli
 Pseudomonas aeruginos
 Staphylococcus aureus
 Salmonella
PREPRATION OF SAMPLING BOTTLES

Sampling of water shall be done by qualified microbiologist.

Sterilize the clean bottle in autoclave at 121°C for 15 minutes.

Dry the bottles from inside so that there should not be any

condensate water inside and then take it to the sampling area.

Add 0.1 ml of 10% sodium thiosulphate solution to every 100 ml water sample
taken for the sampling of chlorinated water sample.

Arrange the required number of suitable sampling glass/plastic bottles in sampling

bin and carry those sampling bottles to the location to be sampled along with other
sampling aids like nitrile gloves ,heat resistance gloves, nose and mouth mask and
70% IPA etc.
SAMPLING OF PURIFIED WATER FOR MICRO TESTING

Take a sterilized 250 ml glass bottle or plastic bottle.

Label each container with specific point code as assigned to each sampling point
,date and time of sampling and sampled by sampling point should be properly
sanitized by 70%IPA to avoid contamination.

Drain the water from sampling points for about 2-3 minutes.

Collect approx 250 ml water purified sample for micro testing from mentioned
user points in point no. 4.3 as per frequency.

Collect sample in appropriate sterile container if it is not possible to test the

sampling within about 2 hours of collection the sample should be held at
refrigerator temperature (2-8°C) for a maximum of about 12 hours to maintain the
microbial attributes until analysis.

If any OOS is observed investigation shall be carried out by microbiologist as per

site SOP no. MC/031.
MICROBIAL TESTING PARAMETERS FOR PURIFIED WATER

Total aerobic plate count (Pour plate method):

Aseptically transfer ,in duplicate ,1ml of water sample to sterile petri dishes.

Add 15-20 ml of sterile R2A media Swirl plates to obtain even dispersion and
allow solidifying.

Inert plates and incubation at 20°C-25°C for 5-7 days.

Quantity the growth by counting all CFUs at the end of incubation period. The
TAPC (cfu/ml) will be arithmetic average of two plates times the dilution factor
( if any). Record results yielding no growth as <1 cfu/ml or as appropriate where
dilutions were performed.

Limit for TAPC results should be <100 cfu/ml.

Perform Gram stain on representive isolate types recovered.

Total coliform count (Membrane Filtration):

Filter approximately 100 ml of test sample through the filter under vacuum.

Aseptically roll filter onto the surface of prepoured petri dish containing M-Endo
agar.

Incubate upright at 30°C - 35°C for 22 – 24 hours.

If no growth observed, indicates absence of coli from. It should be reports as

absent /100 ml.
Test for specified microorganism:

Take 10 ml sample and transfer in 90 ml of sterile soya Aobtain an even dispersion

then take 10 ml of early prepared sample (soluble broth) and incubate it in to100
ml SCDM and incubate at 30-35°C for 18-24 hrs and perform given below
organism analysis.

Escherichia coli:

Transfer 1ml of enriched sample to 100ml of Mac Conkey broth and incubate at 42
-44°C for 24 to 48 hrs. Subcultures on the plate of Mac Conkey agar and incubate
at 32 - 35°C for 18 to 72 hrs. Growth of pink, non – mucoid colonies indicates the
possible presence of E. coli. This should be confirmed by identification test. If no
growth of such type of colonies, or the identification tests are negative indicates
absence of E. coli and the product passes test.

Pseudomonas aeruginosa:

Subculture on a plate of cetrimide agar and incubate at 32-35°C for 18-72 hrs. A
greenish color colony indicates the possibility of presence of pseudomonas
aeruginosa. This should be confirmed by identification test. If there is no growth of
such type of colonies, or identification tests are negative indicats absence of p.
aeruginosa and product passes test.

Staphylococcus aureus:

Subculture on a plate of Mannitol salt agar and incubate at 32-35°C for 18 to 72

hrs. Yellow or white colonies with yellow zones indicate the possibility of
presence of S. aureus. This should be confirmed by identification test. If there is no
growth of such type of colonies, or the identification tests are negative it indicates
absence of S. aureus and product passes the test.
Salmonella:

Aseptically transfer 10 ml of the test water sample into 90 ml SCDM. Mix well
this container to obtain an even dispersion and incubate at 30-35°C for 18-24 hrs.
After incubation period Transfer 0.1 ml of enriched sample to 10 ml of Rapp port
vassiliadis salmonella enrichment broth medium and incubate at 32-35°C for 24-
48 hrs. Subculture on Xylose lysine deoxycholate agar. Incubate at 32-35°C for
24-48 hrs. Growth of well developed, redcolonies, with or without black centre
indicates presence of Salmonella. This should be confirmed identification test. If
there is no growth of such type colonies, or identification tests are negative it
indicates Absence of salmonella and the product passes the test.

Bacteriological analysis

The principal risk associated with water in small-community supplies is that of

infectious disease related to faecal contamination. Hence, as described in Chapter
1, the microbiological examination of drinking-water emphasizes assessment of the
hygienic quality of the supply. This requires the isolation and enumeration of
organisms that indicate the presence of faecal contamination. In certain
circumstances, the same indicator organisms may also be used to assess the
efficiency of drinking-water treatment plants, which is an important element of
quality control. Other microbiological indicators, not necessarily associated with
faecal pollution, may also be used for this purpose. The isolation of specific
pathogens in water should be undertaken only by reference laboratories for
purposes of investigating and controlling outbreaks of disease. Routine isolation in
other circumstances is not practical. Detailed methods for use in bacteriological
analysis are described in Annex 5 (multiple-tube method), Annex 6 (membrane-
filtration method), Annex 7 (onsite testing method), and Annex 8 (presence–
absence test).
Indicator organisms

The properties and significance of the commonly used faecal indicator bacteria are
described in detail in Volume 1; a summary is provided here. Escherichia coli is a
member of the family Enterobacteriaceae, and is characterized by possession of the
enzymes β-galactosidase and β-glucuronidase. It grows at 44–45°C on complex
media, ferments lactose and mannitol with the production of acid and gas, and
produces indole from tryptophan. However, some strains can grow at 37°C but not
at 44–45°C, and some do not produce gas. E. coli does not produce oxidase or
hydrolyse urea. Complete identification of the organism is too complicated for
routine use, but a number of tests have been developed for rapid and reliable
identification. Some of these methods have been standardized at international and
national levels and accepted for routine use; others are still being developed or
evaluated. Escherichia coli is abundant in human and animal faeces; in fresh faeces
it may attain concentrations of 109 per gram. It is found in sewage, treated
effluents, and all natural waters and soils subject to recent faecal contamination,
whether from humans, wild animals, or agricultural activity. Recently, it has been
suggested that E. coli may be present or even multiply in tropical waters not
subject to human faecal pollution. However, even in the remotest regions, faecal
contamination by wild animals, including birds, can never be excluded. Because
animals can transmit pathogens that are infective in humans, the presence of E. coli
or thermotolerant coliform bacteria must not be ignored, because the presumption
remains that the water has been faecally contaminated and that treatment has been
ineffective.
Thermotolerant coliform bacteria

Thermotolerant coliform bacteria are the coliform organisms that are able to
ferment lactose at 44–45°C; the group includes the genus Escherichia and some
species of Klebsiella, Enterobacter, and Citrobacter. Thermotolerant coliforms
other than E. coli may also originate from organically enriched water such as
industrial effluents or from decaying plant materials and soils. For this reason, the
term “faecal” coliforms, although frequently employed, is not correct, and its use
should be discontinued. Regrowth of thermotolerant coliform organisms in the
distribution system is unlikely unless sufficient bacterial nutrients are present,
unsuitable materials are in contact with the treated water, the water temperature is
above 13°C, and there is no free residual chlorine. In most circumstances,
concentrations of thermotolerant coliforms are directly related to that of E. coli.
Their use in assessing water quality is therefore considered acceptable for routine
purposes, but the limitations with regard to specificity should always be borne in
mind when the data are interpreted. If high counts of thermotolerant coliforms are
found in the absence of detectable sanitaryhazards, additional confirmatory tests
specific for E. coli should be carried out. National reference laboratories
developing national standard methods are advised to examine the specificity of the
thermotolerant coliform test for E. coli under local conditions. Because
thermotolerant coliform organisms are readily detected, they have an important
secondary role as indicators of the efficiency of water-treatment processes in
removing faecal bacteria. They may therefore be used in assessing the degree of
treatment necessary for waters of different quality and for defining performance
targets for removal of bacteria.
Coliform organisms (total coliforms)

Coliform organisms have long been recognized as a suitable microbial indicator of

drinking-water quality, largely because they are easy to detect and enumerate in
water. The term “coliform organisms” refers to Gram-negative, rod-shaped
bacteria capable of growth in the presence of bile salts or other surface-active
agents with similar growth-inhibiting properties and able to ferment lactose at 35–
37°C with the production of acid, gas, and aldehyde within 24–48 hours. They are
also oxidase-negative and non-spore-forming and display β-galactosidase activity.
Traditionally, coliform bacteria were regarded as belonging to the genera
Escherichia, Citrobacter, Enterobacter, and Klebsiella. However, as defined by
modern taxonomical methods, the group is heterogeneous. It includes
lactosefermenting bacteria, such as Enterobacter cloacae and Citrobacterfreundii,
which can be found in both faeces and the environment (nutrient-rich waters, soil,
decaying plant material) as well as in drinking-water containing relatively high
concentrations of nutrients, as well as species that are rarely, if ever, found in
faeces and may multiply in relatively good-quality drinking-water, e.g.
Serratiafonticola, Rabnellaaquatilis, and Buttiauxellaagrestis. The existence both of
non-faecal bacteria that fit the definitions of coliform bacteria and of lactose-
negative coliform bacteria limits the applicability of this group as an indicator
of faecal pollution. Coliform bacteria should not be detectable in treated
water supplies and, if found, suggest inadequate treatment, posttreatment
contamination, or excessive nutrients. The coliform test can therefore be used
as an indicator both of treatment efficiency and of the integrity of the
distribution system. Although coliform organisms may not always be directly
related to the presence of faecal contamination or pathogens in drinking-
water, the coliform test is still useful for monitoring the microbial quality of
treated piped water supplies. If there is any doubt, especially when coliform
organisms are found in the absence of thermotolerant coliforms and E. coli,
identification to the species level or analyses for other indicator organisms
may be undertaken to investigate the nature of the contamination. Sanitary
inspections will also be needed.

Coliform organisms (total coliforms) Coliform organisms have long been

recognized as a suitable microbial indicator of drinking-water quality, largely
because they are easy to detect and enumerate in water. The term “coliform
organisms” refers to Gram-negative, rod-shaped bacteria capable of growth
in the presence of bile salts or other surface-active agents with similar growth-
inhibiting properties and able to ferment lactose at 35–37°C with the
production of acid, gas, and aldehyde within 24–48 hours. They are also
oxidase-negative and non-spore-forming and display β-galactosidase activity.
Traditionally, coliform bacteria were regarded as belonging to the genera
Escherichia, Citrobacter, Enterobacter, and Klebsiella. However, as defined
by modern taxonomical methods, the group is heterogeneous. It includes
lactosefermenting bacteria, such as Enterobacter cloacae and
Citrobacterfreundii, which can be found in both faeces and the environment
(nutrient-rich waters, soil, decaying plant material) as well as in drinking-
water containing relatively high concentrations of nutrients, as well as species
that are rarely, if ever, found in faeces and may multiply in relatively good-
quality drinking-water, e.g. Serratiafonticola, Rabnellaaquatilis, and
Buttiauxellaagrestis. The existence both of non-faecal bacteria that fit the
definitions of coliform bacteria and of lactose-negative coliform bacteria limits
the applicability of this group as an indicator of faecal pollution. Coliform
bacteria should not be detectable in treated water supplies and, if found,
suggest inadequate treatment, posttreatment contamination, or excessive
nutrients. The coliform test can therefore be used as an indicator both of
treatment efficiency and of the integrity of the distribution system. Although
coliform organisms may not always be directly related to the presence of
faecal contamination or pathogens in drinking-water, the coliform test is still
useful for monitoring the microbial quality of treated piped water supplies. If
there is any doubt, especially when coliform organisms are found in the
absence of thermotolerant coliforms and E. coli, identification to the species
level or analyses for other indicator organisms may be undertaken to
investigate the nature of the contamination. Sanitary inspections will also be
needed.

Multiple tube method

One of the oldest methods is called the multiple tube method. [3] In this method a
measured sub-sample (perhaps 10 ml) is diluted with 100 ml of sterile growth
medium and an aliquot of 10 ml is then decanted into each of ten tubes. The
remaining 10 ml is then diluted again and the process repeated. At the end of 5
dilutions this produces 50 tubes covering the dilution range of 1:10 through to
1:10000.

The tubes are then incubated at a pre-set temperature for a specified time and at the
end of the process the number of tubes with growth in is counted for each dilution.
Statistical tables are then used to derive the concentration of organisms in the
original sample. This method can be enhanced by using indicator medium which
changes colour when acid forming species are present and by including a tiny
inverted tube called a Durham tube in each sample tube. The Durham inverted tube
catches any gas produced. The production of gas at 37 degrees Celsius is a strong
indication of the presence of Escherichia coli.
The multiple-tube method is also referred to as the most probable
number (MPN) method because—unlike the MF method—it is based
on an indirect assessment of microbial density in the water sample by
reference to statistical tables to determine the most probable number
of microorganisms present in the original sample. It is essential for
highly turbid samples that cannot be analysed by membrane
filtration. The technique iThe multiple-tube method depends on the
separate analysis of a number of volumes of the same sample. Each
volume is mixed with culture medium and incubated. The
concentration of microorganisms in the original sample can then be
estimated from the pattern of positive results (the number of tubes
showing growth in each volume series) by means of statistical tables
that give the “most probable number” per 100ml of the original
sample. The combination of sample volumes for processing is selected
according to the type of water sample or known degree of
contamination. Various configurations and tables may be used;
typical volumes and dilutions are summarized. Appropriate volumes
of water are added aseptically to tubes or other vessels containing
sterile nutrient medium of a concentration that will ensure the
mixture corresponds to single-strength medium. For example, 10ml of
sample would typically be added to 10ml of double-strength medium
or 1ml of sample to 10ml of single-strength medium and so on. The
tube must also contain a small inverted glass tube (Durham tube) to
facilitate the detection of gas production. Growth in the medium is
confirmed by visible turbidity and/or a colour change. Tubes are
incubated without resuscitation, and the number of positive reactions
is recorded after 24 and/or 48 hours, depending on the type of
analysis.used extensively for drinking-water analysis, but it is time-
consuming to perform and requires more equipment, glassware, and
consumables than membrane filtration. However, the multipletube
method may be more sensitive than membrane filtration.

Membrane-filtration method

In the membrane-filtration (MF) method, a minimum volume of 10ml of the

sample (or dilution of the sample) is introduced aseptically into a sterile or
properly disinfected filtration assembly containing a sterile membrane filter
(nominal pore size 0.2 or 0.45µm). A vacuum is applied and the sample is
drawnthrough the membrane filter. All indicator organisms are retained on
or within the filter, which is then transferred to a suitable selective culture
medium in a Petri dish. Following a period of resuscitation, during which the
bacteria become acclimatized to the new conditions, the Petri dish is
transferred to an incubator at the appropriate selective temperature where it
is incubated for a suitable time to allow the replication of the indicator
organisms. Visually identifiable colonies are formed and counted, and the
results are expressed in numbers of “colonyforming units” (CFU) per 100ml
of original sample. This technique is inappropriate for waters with a level of
turbidity that would cause the filter to become blocked before an adequate
volume of water had passed through. When it is necessary to process low
sample volumes (less than 10ml), an adequate volume of sterile diluent must
be used to disperse the sample before filtration and ensure that it passes
evenly across the entire surface of the membrane filter. Membrane filters may
be expensive in some countries. Typical sample volumes for different water
types are shown in Table 4.3. Where the quality of the water is totally
unknown, it may be advisable to test two or more volumes in order to ensure
that the number of colonies on the membrane is in the optimal range for
counting (20–80 colonies per membrane).

Most modern laboratories use a refinement of total plate count in which serial
dilutions of the sample are vacuum filtered through purpose made membrane
filters and these filters are themselves laid on nutrient medium within sealed plates.
[6]
 The methodology is otherwise similar to conventional total plate counts.
Membranes have a printed millimetre grid printed on and can be reliably used to
count the number of colonies under a binocular microscope.

ATP Testing

An ATP test is the process of rapidly measuring active microorganisms in water

through detection adenosine triphosphate (ATP). ATP is a molecule found only in
and around living cells, and as such it gives a direct measure of biological
concentration and health. ATP is quantified by measuring the light produced
through its reaction with the naturally occurring enzyme firefly luciferase using
a luminometer. The amount of light produced is directly proportional to the amount
of biological energy present in the sample.

Second generation ATP tests are specifically designed for water, wastewater and

industrial applications where, for the most part, samples contain a variety of
components that can interfere with the ATP assay.
Plate count

The plate count method relies on bacteria growing a colony on a nutrient medium
so that the colony becomes visible to the naked eye and the number of colonies on
a plate can be counted. To be effective, the dilution of the original sample must be
arranged so that on average between 30 and 300 colonies of the target bacterium
are grown. Fewer than 30 colonies makes the interpretation statistically unsound
whilst greater than 300 colonies often results in overlapping colonies and
imprecision in the count. To ensure that an appropriate number of colonies will be
generated several dilutions are normally cultured. This approach is widely utilised
for the evaluation of the effectiveness of water treatment by the inactivation of
representative microbial contaminants such as E. coli following ASTM D5465.[4][5]

The laboratory procedure involves making serial dilutions of the sample (1:10,
1:100, 1:1000, etc.) in sterile water and cultivating these on nutrient agar in a dish
that is sealed and incubated. Typical media include plate count agar for a general
count or MacConkey agar to count Gram-negative bacteria such as E. coli.
Typically one set of plates is incubated at 22 °C and for 24 hours and a second set
at 37 °C for 24 hours. The composition of the nutrient usually
includes reagents that resist the growth of non-target organisms and make the
target organism easily identified, often by a colour change in the medium. Some
recent methods include a fluorescent agent so that counting of the colonies can be
automated. At the end of the incubation period the colonies are counted by eye, a
procedure that takes a few moments and does not require a microscope as the
colonies are typically a few millimetres across.
Membrane filtration

Most modern laboratories use a refinement of total plate count in which serial
dilutions of the sample are vacuum filtered through purpose made membrane
filters and these filters are themselves laid on nutrient medium within sealed
plates.The methodology is otherwise similar to conventional total plate counts.
Membranes have a printed millimetre grid printed on and can be reliably used to
count the number of colonies under a binocular microscope.

Pour plate method

When the analysis is looking for bacterial species that grow poorly in air, the initial
analysis is done by mixing serial dilutions of the sample in liquid nutrient agar
which is then poured into bottles which are then sealed and laid on their sides to
produce a sloping agar surface. Colonies that develop in the body of the medium
can be counted by eye after incubation.

The total number of colonies is referred to as the total viable count (TVC). The

unit of measurement is cfu/ml (or colony forming units per millilitre) and relates to
the original sample. Calculation of this is a multiple of the counted number of
colonies multiplied by the dilution used.
Physicochemical analysis

Chlorine residual The disinfection of drinking-water supplies constitutes an

important barrier against waterborne diseases. Although various disinfectants may
be used, chlorine in one form or another is the principal disinfecting agent
employed in small communities in most countries. Chlorine has a number of
advantages as a disinfectant, including its relative cheapness, efficacy, and ease of
measurement, both in laboratories and in the field. An important additional
advantage over some other disinfectants is that chlorine leaves a disinfectant
residual that assists in preventing recontamination during distribution, transport,
and household storage of water. The absence of a chlorine residual in the
distribution system may, in certain circumstances, indicate the possibility of post-
treatment contamination. Three types of chlorine residual may be measured: free
chlorine (the most reactive species, i.e. hypochlorous acid and the hypochlorite
ion); combined chlorine (less reactive but more persistent species formed by the
reaction of free chlorine species with organic material and ammonia); and total
chlorine (the sum of the free and combined chlorine residuals). Free chlorine is
unstable in aqueous solution, and the chlorine content of water samples may
decrease rapidly, particularly at warm temperatures. Exposure to strong light or
agitation will accelerate the rate of loss of free chlorine. Water samples should
therefore be analysed for free chlorine immediately on sampling and not stored for
later testing. The method recommended for the analysis of chlorine residual in
drinkingwater employs N,N-diethyl-p-phenylenediamine, more commonly referred
to as DPD. Methods in which o-tolidine is employed were formerly recommended,
but this substance is a recognized carcinogen, and the method is inaccurate and
should not be used. Analysis using starch–potassium iodide is not specific for free
chlorine, but measures directly the total of free and combined chlorine; the method
is not recommended except in countries where it is impossible to obtain or prepare
DPD. Procedures for the determination of free chlorine residual are described in
Annex 9.

pH

It is important to measure pH at the same time as chlorine residual since the

efficacy of disinfection with chlorine is highly pH-dependent: where the pH
exceeds 8.0, disinfection is less effective. To check that the pH is in the
optimalrange for disinfection with chlorine (less than 8.0), simple tests may be
conducted in the field using comparators such as that used for chlorine residual.
With some chlorine comparators, it is possible to measure pH and chlorine residual
simultaneously. Alternatively, portable pH electrodes and meters are available. If
these are used in the laboratory, they must be calibrated against fresh pH standards
at least daily; for field use, they should be calibrated immediately before each test.
Results may be inaccurate if the water has a low buffering capacity. Procedures for
measuring pH using a comparator are described in Annex 10.

Turbidity

Turbidity is important because it affects both the acceptability of water to

consumers, and the selection and efficiency of treatment processes, particularly the
efficiency of disinfection with chlorine since it exerts a chlorine demand and
protects microorganisms and may also stimulate the growth of bacteria. In all
processes in which disinfection is used, the turbidity must always be low—
preferably below 1 NTU or JTU (these units are interchangeable in practice). It is
recommended that, for water to be disinfected, the turbidity should be consistently
less than 5 NTU or JTU and ideally have a median value of less than 1 NTU.
Turbidity may change during sample transit and storage, and should therefore be
measured on site at the time of sampling. This can be done by means of electronic
meters (which are essential for the measurement of turbidities below 5 NTU). For
the monitoring of small-community water supplies, however, high sensitivity is not
essential, and visual methods that employ extinction and are capable of measuring
turbidities of 5 NTU and above are adequate. These rely on robust, low-cost
equipment that does not require batteries and is readily transportable in the field,
and are therefore generally preferred. Procedures for measuring turbidity in the
field using a simple “turbidity tube” are described in Annex 10.

Aesthetic parameters

Aesthetic parameters are those detectable by the senses, namely turbidity, colour,
taste, and odour. They are important in monitoring community water supplies
because they may cause the water supply to be rejected and alternative (possibly
poorer-quality) sources to be adopted, and they are simple and inexpensive to
monitor qualitatively in the field.
Colour

Colour in drinking-water may be due to the presence of coloured organic matter,

e.g. humic substances, metals such as iron and manganese, or highly coloured
industrial wastes. Drinking-water should be colourless. For the purposes
ofsurveillance of community water supplies, it is useful simply to note the presence
or absence of observable colour at the time of sampling. Changes in the colour of
water and the appearance of new colours serve as indicators that further
investigation is needed.

Taste and odour Odours in water are caused mainly by the presence of organic
substances. Some odours are indicative of increased biological activity, others may
result from industrial pollution. Sanitary inspections should always include the
investigation of possible or existing sources of odour, and attempts should always
be made to correct an odour problem. Taste problems (which are sometimes
grouped with odour problems) usually account for the largest single category of
consumer complaints. Generally, the taste buds in the oral cavity detect the
inorganic compounds of metals such as magnesium, calcium, sodium, copper, iron,
and zinc. As water should be free of objectionable taste and odour, it should not be
offensive to the majority of the consumers. If the sampling officer has reason to
suspect the presence of harmful contaminants in the supply, it is advisable to avoid
direct tasting and swallowing of the water. Under these circumstances, a sample
should be taken for investigation to a central laboratory.
Maintenance of Aseptic Condition:

The culture room, incubators, laminar air flow, hot air oven etc. were
fumigated at least once a month with potassium permanganate
(KMnO4) and formaldehyde. Laboratory was kept closed overnight.
Sterility of culture room was monitored by exposing NAA medium
(Nutrient Agar Agar) having composition given in Table 2 and PDA
medium having composition as given in Table 3 (Potato Dextrose
Agar) plated at regular intervals. All the cultures were prepared in
laminar air flow before and after using 70% IPA for wiping.

Cleaning of Glassware:
ALL glassware including culture tubes, beaker measuring cylinder,
reagent bottles, coupling jar, pipette etc. were dipped in 5% chromic
acid (composition in Table 4) and kept it overnight to remove all the
traces of impurities. These were then washed with running water
followed by 2% liquid soap solution (Hi-Media Laboratories,
Mumbai, India). Glassware were again washed with and were dried in
hot air oven.
Sterilization of Glassware:

Reagent bottles, culture vials, beaker and all other glassware which were used in
this study were wrapped with brown paper. Screw caps and rubber corks were
wrapped in a separate beaker which was also covered by brown paper. Micro tip
were kept in their box and wrapped with brown paper separately. All the packed
culture vessels were labeled and sterilized by autoclaving at 15Ibs/inch2 for 15
min.
STAINING:

Gram stain:

This technique is used to a slide such as a fecal smear to observe the

bacterialmicroflora present based on their gram stain reaction.

 Heat-fix the slide with the specimen by passing it over a heat

source, such as a flame, several times using a forceps. The slide
should be passed very quickly through the flame and not be
heated excessively. Place slide on the staining tray.
 Flood the fixed smear with crystal violet solution (#1) and
allow to remain for 1 minute.
 Rinse off the crystal violet with distilled of tap water.
 Flood the slide with iodine solution (#2). Allow to remain for
one minute.
 Rinse off the iodine solution with distilled or tap water.
 Flood the slide with decolorizer (#3) for one to five seconds.
 Rinse off the decolorizer with distilled or tap water.
 Flood the slide with safranin (#4). Allow to remain for 30
seconds.
 Rinse off the safranin with distilled or tap water.
 Dry the slide on bibulous paper or absorbent paper and place in an upright
position. Microscopically examine the slide of bacterial organisms under a 100X
objective. Observe several fields on the slide of bacterial organisms. Describe the
gram reaction of any organisms seen. Gram – positive bacteria stain deep violet to
blue and gram –negative bacteria stain pink to red.
DIGRAMNS

Methodology:

1) MOST PROBABLE NUMBRE (MPN)

 2) Plate Count Method

3) Membrane Filtration Method

 The MF Technique was accepted by the U.S. EPS for microbiology

testing of water in the 11th edition of Standard Methods for the
Examination of Water and Wastewater.
4) Pathogen Detection test

In qualitative estimation the four more commonly found pathogenic bacteria:

1. Escherichia coli
2. Staphylococcus aureus
3. Pseudomonas aeruginosa
4. Salmonella enterica

Initial process:
 Firstly samples are collect
 Now aseptically transfer 10 ml of prepare sample into 100 ml of
Soyabean Casein Digest Media(SCDM).
 Incubate at 35-37°C for 18-48 hours.
 Observe after incubation, if growth is present carry pathogen testing
on selective media.

Pathogen in water

 Pseudomonas aeruginosa
 Staphylococcus aureus
 Salmonella enterica
 Escherichia coli
 Pathogen in water

90 ml SCDM +10 ml sample

Incubate 30-35°C (24 hrs.)

CA media (streak) P.aeruginosa

(3 days -72 hrs.) ,Green MSA (streak) S. aureus (3 days
colonies. -72 hrs.),Yellow colonies.

E. coli (1 ml S.B.+MCB 100 ml) Salmonella (0.1 ml +10 ml RVSB)

incubate 48 hrs. -42-44°C incubate 30-35°C 24 hrs.

Streak on MCA incubate (30-35°C- Streak on XLDA incubate (30-

72 hrs.) pink colonies. 35°C-48 hrs.) , Black
colonies.
RESULT

1) Most Probable Number

 From MPN Index

Water sample Combination of MPN Index per 100ml

positive tubes
Non-potable water 522 170
Potable water 433 44
(Generation point)
Potable water (user 413 28
point)
Purified 000 0
water(Generation point)
Purified water(user 000 0
point)
Water for injection 000 0
(Generation point)
Water for injection 000 0
(User point)
2) Plate count method
Graph (dilution factor v/s number of colonies)

For Generation point For User point

CFU Counting
3) Membrane Filtration Method

4) Pathogen Detection Test

 Escherichia coli
 Staphylococcsaureus

 Pseudomonas aeroginosa
  Salmonella

Discussion
 The results of the water analysis of drinking water from three different water
sources showed that Baddi drinking water was contaminated with coliforms
bacteria. Biochemical tests also showed the existence of coliform bacteria in Baddi
drinking water. Coliform existence was also confirmed by using NAM media when
cultivated at 37C for 24h. In order to analyze the faecal coliforms, samples were
cultured on different biochemical broth and cultivated at 37C for 24 to 48h.
Furthermore, samples were also found to grow on Nutrient agar medium (NAM)
and MacConkey agar medium at 37C. Therefore, gram staining test was carried
out. It confirmed that the bacteria were gram- negative, non-spore forming and rod
shaped. These results confirmed earlier findings. The results reported that the
extracts of neem, Tulsa and garlic have the best antimicrobial activity. The highest
activity recorded against E. coli and the nearest activity recorded against
Enterobacteraerogenes. In the present study, extracts of garlic and neem showed
significant bactericidal activity against E. coli and Enterobacteraerogenes. In
present study, plant extracts showed various levels of antibacterial effect depend
upon the bacterial strains and plant extract concentration. These variations were
found because strains could be genetically different from each other, and this was
probably due to the differences in chemical composition and structure of the cell
wall of both types of microorganisms. Increasing of the concentrations level of
extracts had a significant inhibitory outcome on all studied bacteria. Screening of
bactericidal activity in different ethanolic plant extracts by agar well diffusion test
showed that E. coli and Enterobacteraerogenes were inhibited best in garlic
(6.2mm) followed by Tulsa (3.2 mm) and neem (2 mm) extracts. Gelberg, (1999)
reported in his studies that modification in water quality can be caused by pollution
from both point sources such as industrial and treated sewage releases and diffuse
sources such as storm-water runoff from agricultural and urban areas. Ramesh
(2005) reported that up to 80% of all medical issues in world are due to inadequate
sanitation and polluted water. Chlorination plays as an important factor in removal
of bacteria from water supplies. Chlorine kills microorganisms by cleaving the
chemical bonds in their molecules. Olshansky et al., (1997) reported that
waterborne illnesses are global public health problem and faecal contamination of
potable water imparts an important role in these outbreaks. Water related diseases
present a human tragedy thus killing millions of peoples each year. Cowan, (1999)
studied that plants contained few biologically active chemicals and many of which
had exhibited to have antibacterial properties. Useful antimicrobial compounds in
the plants are categorized into various categories such as phenolic and
polyphenols, terpenoids and fundamental oils, cannabinoids, alkaloids,
lectinsandpolypeptides and polyacetylenes. Sharma, (1981) described that plants
possess various pharmacological properties such as immune-stimulant , tonic,
neurostimulant, anti-aging, anti-bacterial, anti-viral, anti-septic, anti-cancer,
antiasthamatic, anti-inflammatory, antistress properties. Each of three group have
been investigated experimentally viz., Allium Garlic, Neem and Tulsa.
CONCLUSION

Conclusion

Different water samples were sampled from various unit areas and their bacterial
enumeration was done by serial dilution method. The maximum bacterial count
was in Baddi drinking water source. The bacteria isolated and identified were E.
coli and Enterobacteraerogenes. Further role of the test plant extracts for their
antimicrobial activity was also evaluated. Maximum inhibition was recorded in
garlic (6.2mm) followed by Tulsa (3.2 mm) and neem (2 mm) extracts.

From the above survey of information it clearly indicates that it is very important
to remove contaminants from water to make it useful for both household and
industrial purpose. The available data appear to demonstrate the different methods
used in water purification process. This review provides information on,

 Water treatment methods

 Demineralization

 Ultra filtration

 Water for injection

The results of the microbiological analysis of water samples collected in and

around Vijayawada investigated have shown that effluents from industries and
agricultural waste is a major source of environmental pollution through the
discharge of the effluent into the water body. The water quality is directly related
to health and is important for determination of water utility. Assessment of water
quality is a critical factor for assessment of pollution levels. The results from the
present study clearly pointed out that waters from sites-II, IV, V, VI and VII are
highly polluted as they contain high levels of dissolved solids, microbiological
values are not within the permissible limits given by WHO. The waters from
agricultural field’s site-III and IV are contaminated with pesticide residues and
agricultural waste, whereas at sites V, VI and VII from industrial effluents, diesel
from rail engines and domestic wastes respectively. Elevated levels of these
pollution indicators, when compared to the control would invariably affect the
taste, smell, appearance and aesthetic properties of the water or could pose a
potential health hazard of varying degrees to various life forms, which depend on
the water for domestic and recreational purposes. Hence these waters need
conventional treatment including disinfection.
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