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Basic Medical Sciences
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Environmental
&
Pharmaceutical Microbiology

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 Table of Contents
Contents Page No.

1. Introduction - Environmental Microbiology 3 –4

2. Chapter 1 – Air Microbiology 6 – 55

3. Chapter 2 – Microbiology of Sewage 56 – 80

4. Chapter 3 – Aquatic or Microbiology of Water 81 – 117

5. Chapter 4 – Pharmaceutical Microbiology 118 – 138

6. Glossary 139 – 143

7. Reference 144

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Environmental Microbiology
Introduction:

Microorganisms participate in processes that help maintain the

chemical balance and biological composition of natural environments.
The microbial activities involved in the natural cycling of carbon,
nitrogen and sulphur are harnessed in the designs of sewage and
industrial waste water treatment facilities. Microorganisms are also
vital in the preparation and preservation of many foods and beverages.
The product of microbial metabolism are commercially valuable;
without microorganisms there would be no antibiotics and many
amino-acids, vitamins and enzymes would be unavoidable or too
expensive. The 1980s have witnessed an advanced biotechnology in
which breakthroughs in molecular biology have enabled genetic
manipulation of biologic systems. This technology has the potential to
significantly alter the relationship between humankind and all the rest
of the living world, on a scale so vast that its dimensions are only
beginning to be understood.

Environmental Microbiology:

The dynamic interaction of the microbes with the physical and

chemical make up of world’s many ecosystems is the subject of
environmental microbiology. The environment is the natural
surroundings around us. Habitat is the physical space or location where
a species live.

Microbial populations play an essential role in maintaining an

ecological balance between the biomass and the available nutrients.
Our natural environment is taken for granted by most of us, until it is
altered in same way, as by overuse or pollution. Preservation of a
natural environment is heavily dependent on the role microbes play in
maintaining the chemical balance between available nutrients and in
metabolizing waste products. Pollutants most often the chemical

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residues of human activities can disrupt this balance and cause havoc to
the environment.

Ecosystem:

An Ecosystem is the total community of organisms in a physically

defined space. Ecosystems may be
1. Small, eg. swamps, ponds, streams or
2. Huge eg. The Chesapeake Bay Estuary or one of the Great
Lakes.

An ecosystem is confined by geological formations – the shores of

the pond, the banks of the river, the bottom of a Bay or Lake.
Within this confined space a chemical environment that greatly
influences both the quantity and composition of its biota. Biota is
the sum total of the living organisms in any designated area. These
physical and chemical characteristics are affected by rains that
swell rivers or streams by tides that cause cyclical changes in the
oceans and estuaries by the addition of nutrients from runoffs,
industrial waste and sewage and so on.

Environmental Microbiology as already defined is the dynamic

interactions of the microbes with the physical and chemical make
up of the world’s many ecosystems are the subject of
environmental microbiology. Microbial ecology is the study of
the relationships between populations of microorganisms and their
environments. Each ecosystem is composed of many microbial
communities made distinct by local physical and chemical
parameters.

1. Temperature,
2. pH and
3. Availability of oxygen and nutrients dictate the species
and numbers of microbes in these communities.

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The physical space or location where a species lives is its habitat
and every microorganisms must have atleast one natural habitat,
otherwise cannot survive the rigors of its world.

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 Chapter 1

AIR MICROBIOLOGY
Air Microbiology & Air Sanitation

Contents:

1. Number and kind of organisms in Air, Distribution

and sources of airborne organisms; Droplet and
Droplet Nuclei.
2. Enumeration of microorganisms in Air – Air
Samplers and Sampling Techniques.
3. Potential Hazards of Laboratory Techniques
4. Air-borne diseases, significance of air flora in
human health.
5. Air Sanitation – U.V.Light, Gaseous agents
6. Laminar Air Flow

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INTRODUCTION

AIR

Of all environments, air is the simplest one and it occurs in a single

phase gas. The relative quantities of various gases in air, by volume
percentage are

1. Nitrogen 78%,
2. Oxygen 21 %,
3. Argon 0.9%,
4. Carbon dioxide 0.03%,
5. Hydrogen 0.01 % and
6. Other gases in trace amounts.
7. In addition to various gases, dust and condensed vapour may also
be found in air.

Various layers can be recognized in the atmosphere upto a height of

about 1000km. The layer nearest to the earth is called as troposphere.
In temperate regions, troposphere extends upto about 11 km whereas in
tropics up to about 16km. This troposphere is characterized by a heavy
load of microorganisms.

The temperature of the atmosphere varies near the earth's surface.

However, there is a steady decrease of about 1˚C per 150m until the top
of the troposphere. Above the troposphere, the temperature starts to
increase.

The atmosphere as a habitat is characterised by high light intensities,

extreme temperature variations, low amount of organic matter and a
scarcity of available water making it a non hospitable environment
for microorganisms and generally unsuitable habitat for their
growth. Nevertheless, substantial number of microbes is found in the
lower regions of the atmosphere

In the course of a day a man requires about 500 cc. ft. of air. Hence the
bacterial content of the air he breaths is important, particularly so, when
it contains pathogens and that too in significant numbers likely to cause

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disease. No organisms are natural inhabitants of the atmosphere, nor
do microorganisms multiply in the air because air does not contain the
necessary amount of moisture and utilizable form of nutrients.

Microbes Found in Air- In addition to gases, dust particles and water

vapour, air also contains microorganisms. There are vegetative cells
and spores of bacteria, fungi and algae, viruses and protozoan cysts.
Since air is often exposed to sunlight, it has a higher temperature and
less moisture. So, if not protected from desiccation, most of these
microbial forms will die.

Air is mainly a transport or dispersal medium for microorganisms.

Microorganisms occur in relatively small numbers in air when
compared with soil or water. The microflora of air can be studied under
two headings outdoor and indoor microflora.

Sources of Microorganisms in Air - Although a number of

microorganisms are present in air, it doesn't have an indigenous flora.
Air is not a natural environment for microorganisms as it doesn't
contain enough moisture and nutrients to support their growth and
reproduction.

The microorganisms found in air are mainly derived from the

1. soil
2. natural bodies of water
3. plants
4. man and animals

Quite a number of sources have been studied in this connection and

almost all of them have been found to be responsible for the air
microflora. One of the most common sources of air microflora is the
soil.

Soil microorganisms when disturbed by the wind blow, liberated into

the air and remain suspended there for a long period of time. Man made
actions like digging or ploughing the soil may also release soilborne
microbes into the air. Similarly microorganisms found in water may

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also be released into the air in the form of water droplets or aerosols.
Splashing of water by wind action or tidal action may also produce
droplets or aerosols.

Air currents may bring the microorganisms from plant or animal

surfaces into air. These organisms may be either commensals or plant
or animal pathogens. Studies show that plant pathogenic
microorganisms are spread over very long distances through air. For
example, spores of Puccinia graminis travel over a thousand kilometers.
However, the transmission of animal diseases is not usually important
in outside air.

The main source of airborne microorganisms is human beings. Their

surface flora may be shed at times and may be disseminated into the
air. Similarly, the commensal as well as pathogenic flora of the upper
respiratory tract and the mouth are constantly discharged into the air by
activities like coughing, sneezing, talking and laughing.

The microorganisms are discharged out in three different forms which

are grouped on the basis of their relative size and moisture content.
They are droplets, droplet nuclei and infectious dust. It was Wells,
who described the formation of droplet nuclei. This initiated the studies
on the significance of airborne transmission. A brief description of
these agents is given below.

Droplets - Droplets are usually formed by sneezing, coughing or

talking. Each consists of saliva and mucus. Droplets may also contain
hundreds of microorganisms which may be pathogenic if discharged
from diseased persons. Pathogens will be mostly of respiratory tract
origin. The size of the droplet determines the time period during which
they can remain suspended.

Most droplets are relatively large, and they tend to settle rapidly in still
air. When inhaled these droplets are trapped on the moist surfaces of
the respiratory tract. Thus, the droplets containing pathogenic
microorganisms may be a source of infectious disease.

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Droplet Nuclei - Small droplets in a warm, dry atmosphere tend to
evaporate rapidly and become droplet nuclei. Thus, the residue of solid
material left after drying up of a droplet is known as droplet nuclei.
These are small, 1-4µm, and light. They can remain suspended in air
for hours or days, traveling long distances.

They may serve as a continuing source of infection if the bacteria

remain viable when dry. Viability is determined by a set of complex
factors including, the atmospheric conditions like humidity, sunlight
and temperature, the size of the particles bearing the organisms, and the
degree of susceptibility or resistance of the particular microbial species
to the new physical environment.

If inhaled droplet nuclei tend to escape the mechanical traps of the

upper respiratory tract and enter the lungs. Thus, droplet nuclei may act
as more potential agents of infectious diseases than droplets.

Infectious Dust - Large aerosol droplets settle out rapidly from air on
to various surfaces and get dried. Nasal and throat discharges from a
patient can also contaminate surfaces and become dry. Disturbance of
this dried material by bed making, handling a handkerchief having
dried secretions or sweeping floors in the patient's room can generate
dust particles which add microorganisms to the circulating air.

Microorganisms can survive for relatively longer periods in dust. This

creates a significant hazard, especially in hospital areas. Infective dust
can also be produced during laboratory practices like opening the
containers of freeze dried cultures or withdrawal of cotton plugs that
have dried after being wetted by culture fluids. These pose a threat to
the people working in laboratories

Significance of Air Microflora - Although, when compared with the

microorganisms of other environments, air microflora are very low in
number, they play a very significant role. This is due to the fact that the
air is in contact with almost all animate and inanimate objects.

The significance of air flora has been studied since 1799, in which year
Lazaro Spallanzani attempted to disprove spontaneous generation. In

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the year 1837, Theodore Schwann, in his experiment to support the
view of Spallanzani, introduced fresh heated air into a sterilized meat
broth and demonstrated that microbial growth couldn't occur.

This formed the basis of modern-day forced aeration fermentations. It

was Pasteur in 1861, which first showed that microorganisms could
occur as airborne contaminants. He used special cotton in his air
sampler onto which the microorganisms were deposited.

He microscopically demonstrated the presence of microorganisms in

the cotton. In his famous swan necked flask experiment, he showed that
growth could not occur in sterile media unless airborne contamination
had occurred

Factors Affecting Air Microflora - A number of intrinsic and

environmental factors influences the kinds and distribution of the
microflora in air. Intrinsic factors include the nature and physiological
state of microorganisms and also the state of suspension. Spores are
relatively more abundant than the vegetative bacterial cells.

This is mainly due to the dormant nature of spores which enables

them to tolerate unfavourable conditions like desiccation, lack of
enough nutrients and ultraviolet radiation. Similarly fungal spores are
abundant in the air since they are meant for the dispersal of fungi.

The size of the microorganisms is another factor that determines the

period of time for which they remain suspended in air. Generally
smaller microorganisms are easily liberated into the air and remain
there for longer period. Fungal mycelia have a larger size and hence
mainly fragments of mycelia will be present in air. The state of
suspension plays an important role in the settling of microorganisms in
air. Organisms in the Free State are slightly heavier than air and settle
out slowly in a quiet atmosphere. However, microorganisms suspended
in air are only rarely found in the free state.

Usually they are attached to dust particles and saliva. Microorganisms

embedded in dust particle settle out rapidly and in a quiet atmosphere
they remain airborne only for a short period of time. Droplets which are

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discharged into the air by coughing or sneezing are also remain
suspended in air for a short period of time. When their size decreases
by evaporation they remain for a longer period in air.

Environmental factors that affect air microflora include

1. atmospheric temperature,
2. humidity, air current,
3. The height at which the microorganisms are found etc.
4. Temperature and relative humidity are the two important factors
that determine the viability of microorganisms in aerosol.

Studies with Serratia marcesens and E. coli show that the airborne
survival is closely related to the temperature. There is a progressive
increase in the death rate with an increase in temperature from -18°C to
49°C. Viruses in aerosols show a similar behaviour. Particles of
influenza, poliomyelitis and vaccinia viruses survive better at low
temperature from 7 to 24°C. The optimum rate of relative humidity
(RH) for the survival of most microorganisms is between 40 and 80
percent. Low and high relative humidity cause the death of most
microorganisms. Almost all viruses survive better at a RH of 17 to 25
percent.

A notable exception is that of poliomyelitis which survives better at 80

to 81 percent. Survival has been found to be a function of both RH and
temperature. At all temperatures, survival is best at the extremes of RH.
Irrespective of RH, an increase in temperature leads to decrease in
survival time.

Air current influences the time for which either the microorganisms or
the particles laden with microorganisms remain suspended in air. In
still air the particles tend to settle down. But a gentle air current can
keep them in suspension for relatively long periods. Air current is also
important in the dispersal of microorganisms as it carries them over a
long distance.

Air currents also produce turbulence which causes a vertical

distribution of air flora. Global weather patterns also influence the

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vertical distribution. High altitudes have a limiting effect on the air
microflora. High altitudes are characterized by severe conditions like
desiccation, ultraviolet radiation and low temperature. Only resistant
forms like spores can survive these conditions. Thus high altitudes are
characterized by the presence of spores and other resistant forms.

Distribution of Microbes in Air - No microbes are indigenous to the

atmosphere rather they represent allochthonous populations transported
from aquatic and terrestrial habitats into the atmosphere. Microbes of
air within 300-1,000 or more feet of the earth's surface are the
organisms of soil that have become attached to fragments of dried
leaves, straw or dust particles, being blown away by the wind. Species
vary greatly in their sensitivity to a given value of relative humidity,
temperature and radiation exposures.

More microbes are found in air over land masses than far at sea. Spores
of fungi, especially Alternaria, Cladosporium, Penicillium and
Aspergillus are more numerous than other forms over sea within about
400 miles of land in both polar and tropical air masses at all altitudes
up to about 10,000 feet.

Microbes found in air over populated land areas below altitude of 500
feet in clear weather include spores of Bacillus and Clostridium, ascos-
pores of yeasts, fragments of myceilium and spores of molds and
streptomycetaceae, pollen, protozoan cysts, algae, Micrococcus,
Corynebacterium etc.

In the dust and air of schools and hospital wards or the rooms of
persons suffering from infectious diseases, microbes such as tubercle
bacilli, streptococci, pneumococci and staphylococci have been
demonstrated.

These respiratory bacteria are dispersed in air in the droplets of saliva

and mucus produced by coughing, sneezing, talking and laughing.
Viruses of respiratory tract and some enteric tract are also transmitted
by dust and air. Pathogens in dust are primarily derived from the
objects contaminated with infectious secretions that after drying
become infectious dust.

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Droplets are usually formed by sneezing, coughing and talking. Each
droplet consists of saliva and mucus and each may contain thousands of
microbes. It has been estimated that the number of bacteria in a single
sneeze may be between 10,000 and 100,000. Small droplets in a warm,
dry atmosphere are dry before they reach the floor and thus quickly
become droplet nuclei.

Many plant pathogens are also transported from one field to another
through air and the spread of many fungal diseases of plants can be
predicted by measuring the concentration of airborne fungal spores.
Human bacterial pathogens which cause important airborne diseases
such as diphtheria, meningitis, pneumonia, tuberculosis and whooping
cough are described in the chapter "Bacterial Diseases of Man".

Air Microflora Significance in Hospitals - Although hospitals are the

war fields for combating against diseases, there are certain occasions in
which additional new infectious diseases can be acquired during
hospitalization. Air within the hospital may act as a reservoir of
pathogenic microorganisms which are transmitted by the patients and is
commonly referred to as Indoor Air.

Infection acquired during the hospitalization is called nosocomial

infections and the pathogens involved are called as nosocomial
pathogens. Infections, manifested by the corresponding symptoms,
after three days of hospitalization can be regarded as nosocomial
infection according to Gleckman & Hibert, 1982 and Bonten&
Stobberingh, 1995.

Nosocomial infection may arise in a hospital unit or may be brought in

by the staff or patients admitted to the hospital. The common
microorganisms associated with hospital infection are

1. Haemophilus influenzae,
2. Streptococcus pneumoniae,
3. Staphylococcus aureus,
4. Pseudomonas aeruginosa,
5. members of Enterobacteriaceae and
6. Respiratory viruses.

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Development of high antibiotic resistance is a potential problem among
nosocomial pathogens. For example, Methicillin Resistant
Staphylococcus aureus (MRSA) and gentamicin resistant Gram-
negative bacilli are of common occurrence. Even antiseptic liquids used
would contain bacteria, for example Pseudomonas, due to their natural
resistance to certain disinfectants and antiseptics and to many other
antibiotics.

Nosocomial pathogens may cause or spread hospital outbreaks.

Nosocomial pneumonia is becoming a serious problem nowadays and
a number of pathogens have been associated with it (Bonten &
Stobberingh, 1995). Frequent agents are Staphylococcus aureus,
Streptococcus pneumoniae, Pseudomonas aeruginosa, Enterobacter,
Klebsiella, Escherichia coli and Haemophilus influenzae. Other less
frequent agents are enterococci, streptococci other than S. pneumoniae,
Serratia marcescens, Citrobacter freundii, Acinetobacter sp. and
Xanthomonas sp.

In addition Legionella, Chlamydia pneumoniae and Mycobacterium

tuberculosis have also been reported. Nosocomial transmissions of
tuberculosis from patients to patients and from patients to health care
workers have also been well documented (Wenger et a/., 1995).

There are two main routes of transmission for nosocomial pathogens,

contact (either direct or indirect) and airborne spread. Airborne spread
is less common than the spread by direct or indirect contact. It occurs
by the following mechanisms. The source may be either from persons
or from inanimate objects.

In case of spread from persons the droplets from mouth, skin scales
from nose, skin exudates and infected lesion transmit diseases such as
measles, tuberculosis, pneumonia, staphylococcal sepsis and
streptococcal sepsis. Talking, coughing and sneezing produce droplets.
Skin scales are shed during wound dressing or bed making.

In case of inanimate sources particles from respiratory equipment and

air-conditioning plant may transmit diseases. These include Gram-

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negative respiratory infection, Legionnaire's disease and fungal
infections.

Air Microflora Significance in Human Health - The significance of

air microflora in human health relies on the fact that air acts as a
medium for the transmission of infectious agents. An adult man inhales
about '5m3 of air per day. Although most of the microorganisms
present in air are harmless saprophytes and commensals, less than 1%
of the airborne bacteriae are pathogens.

Eventhough the contamination level is very low the probability of a

person becoming infected will be greatest if he is exposed to a high
concentration of airborne pathogens. Carriers, either with the
manifestation of corresponding symptoms or without any apparent
symptoms, may continuously release respiratory pathogens in the
exhaled air.

Staphylococcus aureus is the most commonly found pathogen in air

since the carriers are commonly present. The number of S. aureus in air
may vary between 0-l/m3 and 50/m3.

Practically speaking, Outdoor air doesn't contain disease causing

pathogen in a significant number to cause any infection. The purity of
outdoor air, however, is an essential part of man's environment.
Dispersion and dilution by large volume of air is an inherent
mechanism of air sanitation in outside air.

In the case of indoor air chance for the spread of infectious disease is
more, especially in areas where people gather in large numbers. For
example, in theatres, schools etc.

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Significance of Microorganisms in Air - As long as microorganisms
remain in the air they are of little importance. When they come to rest
they may develop and become beneficial or harmful. Knowledge of the
microorganisms in air is of importance in several aspects.

Food manufacture: Microorganisms that have been transported

through the air and have settled on or in the material are involved in
various fermentation products. Production of alcoholic beverages,
vinegar, sauerkraut, ensilage, dairy products, etc., is often due to
microbial activity.

Spoilage of foods and fermentation products: Microorganisms are

often troublesome in the home and in industry where foods and other
fermentation products are prepared. In industrial processes, where
particular organisms are to be grown, to supply sterile air free from
contaminating organisms is a considerable problem.

Airborne diseases: There are two main sources of microorganisms in

air. These are saprophytic soil organisms raised as dust, and
organisms from body tissues introduced into the air during coughing,
sneezing talking, and singing. Most dust particles laden with
microorganisms are relatively large and tend to settle rapidly. Droplets
expelled during coughing, sneezing, etc consist of sativa and mucus,
and each of them may contain thousands of microorganisms.

Most droplets are large, and, like dust, tend to settle rapidly. Some
droplets are of such size that complete evaporation occurs in a warm,
dry climate, and before they reach the floor quickly become droplet
nuclei. These are small and light, and may float about for a relatively
long period.

Airborne diseases are transmitted by two types of droplets, depending

upon their size. (I) Droplet infection proper applies to, droplets larger
than 100 µm in diameter. (2) The other type may be called airborne
infection, and applies to dried residues of droplets. Droplet infection
remains localized and concentrated, whereas airborne infection may be
carried long distances arid is dilute

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Major Diseases Transmitted By Air - Lists the major diseases
transmitted via air. Since air enters the body through the respiratory
tract and since such diseases frequently localize in the nose and throat,
they are called respiratory diseases as a group.

1. Diphtheria, Septic
2. sorethroat,
3. Scarlet fever,
4. Rheumatic fever.
1.Bacterial 5. Tuberculosis,
6. pneumonia,
7. Meningitis,
8. Whooping cough.

1. Smallpox,
2. Chickenpox,
3. Measles,
4. German Measles,
2.Viral
5. Mumps,
6. influenzea
7. Common cold,
8. Psittacosis.
3.Fungal 1. Systemic mycoses.

Enumeration of Microorganisms in Air:

There are several methods, which require special devices, designed for
the enumeration of microorganisms in air. The most important ones are

• solid and liquid impingement devices,

• filtration,
• sedimentation,
• centrifugation,
• Electrostatic precipitation etc.

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However, none of these devices collects and counts all the
microorganisms in the air- sample tested. Some microbial cells are
destroyed and some entirely pass through in all the processes.

Some of the methods are described below.

Impingement in liquids:
In this method, the air is drawn through a very small opening or a
capillary tube and bubbled through the liquid. The organisms get
trapped in the liquid medium. Aliquots of the liquid are then plated to
determine its microbial content. Aliquots of the broth are then plated to
determine microbial content e.g. Bead-bubbler device which is
discussed in detail below.

Impingement on solids:
In this method, the microorganisms are collected, or impinged directly
on the solid surface of agar medium. Colonies develop on the medium
where the organism impinges. Several devices are used, of which the
settling-plate technique is the simplest. In this method the cover of the
pertridish containing an. agar medium is removed, and the agar surface
is exposed to the air for several minutes. A certain number of colonies
develop on incubation of the petridish.

Each colony represents particle carrying microorganisms. Since the

technique does not record the volume of air actually sampled, it gives
only a rough estimate. However, it does give information about the
kind of microorganisms in a particular area. Techniques wherein a
measured. Volume of air is sampled have also been developed. These
are sieve and slit type devices. A sieve device has a large number of
small holes in a metal cover, under which is located a petridish
containing an agar medium.

A measured volume of air is drawn, through these small holes.

Airborne particles impinge upon the agar surface. The plates are
incubated and the colonies counted. In a slit device the air is drawn
through a very narrow slit onto a petridish containing agar medium.

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The slit is approximately the length of the petridish. The petridish is
rotated at a particular speed under the slit One complete turn is made
during the sampling operation.

Filtration:
The membrane filter devices are adaptable to direct collection
of microorganisms by filtration of air. The method is similar in
principle to that described for water sampling.
Hess's Tube Method
This sampler is made of a horizontal glass tube which contains a layer
of solid medium at the bottom. As air is drawn in to the tube through
the inlet end, the particles settle onto the medium. Upon incubation
colonies develop on the medium. If the tube is long enough or the flow
is sufficiently slow, almost all particles will settle out before reaching
the outlet end.

Settle Plate Method

The principle behind this method is that the bacteria carrying particles
are allowed to settle onto the medium for a given period of time and
incubated at the required temperature. A count of colonies formed
shows the number of settled bacteria containing particles.

In this method petridishes containing an agar medium of known surface

area are selected so that the agar surface is dry without any moisture.
Choice of the medium depends upon the kind of microorganisms to be
enumerated. For an overall count of pathogenic, commensal and
saprophytic bacteria in air blood agar can be used.

For detecting a particular pathogen which may be present in only small

numbers, an appropriate selective medium may be used. Malt extract
agar can be used for molds. The plates are labelled appropriately about
the place and time of sampling, duration of exposure etc. Then the
plates are uncovered in the selected position for the required period of
time.

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The optimal duration of exposure should give a significant and readily
countable number of well isolated colonies, for example about 30-100
colonies. Usually it depends on the dustiness of air being sampled. In
occupied rooms and hospital wards the time would generally be
between 10 to 60 'minutes.

During sampling it is better to keep the plates about 1 metre above the
ground. Immediately after exposure for the given period of time, the
plates are closed with the lids. Then the plates are incubated for 24 hrs
at 37°C for aerobic bacteria and for 3 days at 22°C for saprophytic
bacteria.

For molds incubation temperature varies from 1O-50°C for 1-2 weeks.
After incubation the colonies on each plate are counted and recorded as
the number of bacteria carrying particles settling on a given area in a
given period of time.

Though the method has the advantage of simplicity, it has certain

limits. In this method only the rate of deposition of large particles from
the air, not the total number of bacteria carrying particles per volume, is
measured.

Growth of bacteria in the settled particles may be affected by the

medium used since not all microorganisms are growing well on all
media. Moreover since air currents and any temporary disturbances in
the sampling area can affect the count, many plates have to be used.

Air Centrifuge
The first primitive type of air centrifuge was developed by Wells
(1993). The principle of air centrifuge is that the particles from air are
centrifuged onto the culture medium. In his air centrifuge sampled air
was passed along a tube which was rotated rapidly on its long axis. The
inner surface of the tube was lined with culture medium and any
bacteria containing particle deposited on it grew into a colony on
incubation.

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A modern version of this centrifuge is the Reuter centrifugal air
sampler, which is portable and battery powered. It resembles a large
cylindrical torch with an open ended drum at one end. The drum
encloses impeller blades which can be rotated by battery power when
switched on. A plastic strip coated with culture medium can be inserted
along the inner side of the drum.

Air is drawn into the drum and subjected to centrifugal acceleration.

This causes the suspended particles to impact on the culture medium.
After sampling the strip is removed from the instrument and incubated
at 37°C for 48 hours. Later the colonies can be counted.

Advantage of this sampler is that it is very convenient for transportation

and use. The disadvantage is that it is less efficient than the slit sampler
in detecting particle below 5mm in diameter. More over the size of the
air being sampled cannot be accurately controlled.

Filtration
This is a simple method for collecting particles from air. The filter can
be made of any fibrous or granular material like sand, glass fibre and
alginate wool (in phosphate buffer). However, recovery of organisms
for culture is not so easy.

Tube Sampler
This is one of the oldest devices for collecting and enumerating
microorganisms in the air. It consists of a tube with an inlet at the top
and an outlet at the bottom which is narrower than the top end. Near the
bottom there is a filter of wet sand which is supported by a cotton plug
below. The entire device can be sterilized.

After sterilization the air to be sampled is allowed to pass through the

sand and cotton. Microorganisms as well as dust particles containing
microorganisms in the air are deposited in the sand filter as the air
passes through it. Later the sand is washed with broth and a plate count
is made from the broth by taking aliquots of the broth.

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Millipore Filter:
This type of filters is made of pure and biologically inert cellulose
ethers. They are prepared as thin porous, circular membranes of about
150 µm thickness. The filters have different porosity. Grades from
10nm to 811m. The assemblage contains a funnel shaped inlet and a
tube like outlet. In between these two the filter is fitted.

The outlet may be connected to a vacuum pump to suck known amount

of air. After collecting required volume of air through the filter, it can
directly be placed onto the surface of a solid medium. After incubation
colonies formed can be counted.

Impingement on to Liquid:
Impingement on to Liquid is divided into three types, they are
following:

(i) Raised Impinger

(ii) Bead- Buffer Device

(iii) Lemon Sampler

Raised Impinger:
In this type of sampler impingement is made within bulk fluid by a jet of air

Bead Bubbler Device:

It is also an oldest device for sampling air. It consists of a 250ml
suction flask which has an outlet on the side connected to suction
pump. A glass bubbler, which is nothing but a glass tube with minute
openings at the bottom, is kept in place inside the flask by a rubber
stopper. Glass beads of size about 5mm in diameter are kept around the
glass bubbler.

In addition the flask contains known volume of broth. Air is drawn into
the flask through the glass bubbler when the flask is continuously

23
shaken. The incoming air escapes into the broth in the form of bubbles
through the holes at the bottom of glass bubbler.

The shaking action of the flask and hence the glass beads facilitate the
formation of bubbles. Thus, microorganisms in the air are dispersed in
the broth and after sampling an aliquot from the broth is plated for
count.

24
Lemon Sampler:
It consists of a glass Folin aeration tube with a perforated bulb with six
holes at one end. The bulb end of aeration tube is contained within a
test tube by a two hole rubber stopper. The bulb is actually centered
near the bottom of the tube and is immersed in 20ml of broth. Two or
three drops of olive oil is added to the broth to prevent foaming.

A kjeldahl trap with square glass baffle is shortened at both ends for
convenience. The intake end is slightly bent and inserted into the other
hole of the stopper. A flow meter measures the rate of airflow entering
the upper open end of the Folin tube. An air pump is attached to the
exhaust end of the kjeldahl trap.

The entire bubbler should be sterilized by autoclaving. Alternatively it

can be sterilized by rinsing with 70% alcohol and dried afterwards. Air
is drawn at the rate of 25-30 liters per minute and dispersed through the
broth.

Impingement onto Solids:

They are divided into two types

(i)Hollaender and Dalla Valle Sampler

(ii)Slit Sampler

25
Hollaender and Dalla Valle Sampler:
This sampler consists of a brass container with removable bottom. The
container is fitted with an inverted glass funnel. In the lower portion of
container a petridish base with medium is placed which can be screwed
tightly against the gasket during sampling.

The funnel is kept just above the petridish without touching it. The
inside of the funnel and rim are swabbed with alcohol before use. The
air sample passes through the funnel stem and the airborne
microorganisms are impinged upon the agar medium.

The air is drawn by means of a pump in series with a flow meter.

Effective sampling rate is found to be 28 liters per minute. The method
is simple, portable and efficient

Slit Sampler:
Slit sampler is an efficient and convenient device for the enumeration
of bacteria carrying particles in a unit volume of air. It was introduced
by Bourdillon et al. in 1941. It works on the principle that when air is
drawn from the environment at a fixed rate and the suspended particles
are allowed to impinge on the surface of an agar plate on incubation
each particle will form a colony.

The equipment consists of a box closed by an air tight door. There is a

slit of 0.33 mm width and 27.5 mm length with vertical parallel sides of
about 3mm deep at the top through which the sampled air enters. The
box is connected to a suction pump which maintains it at a negative
pressure of 22.6 mm mercury. At the correct negative pressure air will
enter through a slit of above dimension at the rate of one cubic foot
(28.3 lit.) per minute.

Inside the box at the bottom there is a rotating circular platform for
keeping the agar plate. The platform is usually covered with adhesive
or gripping material to ensure the agar plate being rotated with the

26
platform and is not slipping out of position while rotating. When the
agar plate is correctly positioned on the platform the slit will be exactly
2mm above and along the radius of the plate. Thus when the plate is
rotated along with platform

Slit Sampling Procedure:

Agar plates with dry, even surface are selected and marked to record
the sampling area, time of sampling, duration of exposure, volume of
sampled air etc. The slit should be unblocked and free from dust and if
necessary may be cleaned with alcohol and by inserting the edge of a
stiff paper. The door of the box is opened and the plate is placed at the
centre on the platform.

The distance between the agar surface and the slit is adjusted to be
2mm. At the correct time, the motor that rotates the plate and the
suction pump that evacuates the sampler are switched on and the
negative pressure is maintained at -22.6 mm mercury. After sampling
for the time necessary to collect the required volume of air the suction
pump and the rotor are switched off.

The door is opened and the plate is taken out. The plate is covered with
the lid immediately and incubated at 37°C for 24 - 48 hours. After
incubation the colonies are counted and the result is expressed as the
number of bacteria carrying particles per given volume of air.

Limitations of Slit Sampling:

When sitting and operating the sampler inside a room major source of
bacterial contamination in the air is the dust from the skin and clothings
of the operating person liberated by the body, movements. Therefore
unnecessary movements should be avoided while sampling

Advantages of Slit Sampler:

Highly efficient device and can collect upto about 95% of the water
droplet particles sprayed into air. Even respiratory secretion droplet
nuclei of 0.2µm diameter can be collected in large numbers

27
Disadvantages of Slit Sampler:
Slit sampler is cumbersome and noisy equipment. When the vacuum
pump is enclosed with acoustic insulation noise can be reduced to some
extent. Quieter and portable samplers are also available but they are
less efficient in collecting sample particles

Sieve Sampler:
This is a mechanically simpler form of impinger. The instrument is
more or less similar to that of slit sampler with an enclosed chamber.
The particles containing microorganisms are distributed over the plate
as separate air jets through several holes. Upon incubation these
particles form colonies which can be counted.

For more efficient sampling and size grading of particles Anderson

developed a multistage sieve device in which several impingers with
holes of different sizes are arranged in series.

Electrostatic Precipitation:
Electrostatic precipitation is an efficient method of removing particles
from air. In Litton large volume air sampler the air is allowed to pass
through the electrodes.

The charged particles fall on a rotating disc which is fed with collecting
fluid at a rate of 10ml per, minute. Air is sucked into the chamber by a
rotating fan at the bottom. The low resistance of the system enables
high rates of air flow. They are suitable for large volumes of air.

Luckiesh et al. devised a sampler which contains two removable

covers. Each unit has one upper electrode and one lower electrode. In
one unit the upper electrode is negative and the lower electrode is
positive and in the other unit the electrical condition is reversed. Air is
drawn at equal rates in both the units. Charged microorganisms are
collected in the petridishes placed on opposite electrodes.

28
HUMAN LUNGS

In humans the lungs occupy a large portion of the chest cavity from the
collarbone down to the diaphragm. The right lung is divided into three
sections, or lobes. The left lung, with a cleft to accommodate the heart,
has only two lobes. The two branches of the trachea, called bronchi,
subdivide within the lobes into smaller and smaller air vessels known
as bronchioles. Bronchioles terminate in alveoli, tiny air sacs
surrounded by capillaries. When the alveoli inflate with inhaled air,
oxygen diffuses into the blood in the capillaries to be pumped by the
heart to the tissues of the body. At the same time carbon dioxide
diffuses out of the blood into the lungs, where it is exhaled.

In humans and other animals, the respiratory system can be

conveniently subdivided into an upper respiratory tract (or conducting
zone) and lower respiratory tract (respiratory zone), trachea and lungs.
Air moves through the body in the following order:

• Nostrils
• Nasal cavity
• Pharynx (naso-, oro-, laryngo-)
• Larynx (voice box)

29
 • Trachea (wind pipe)
• Thoracic cavity (chest)
• Bronchi (right and left)
• Alveoli (site of gas exchange)

Upper respiratory tract/conducting zone

The conducting zone starts with the nares (nostrils) of the nose, which
open into the nasopharynx (nasal cavity). The primary functions of the
nasal passages are to: 1) filter, 2) warm, 3) moisten, and 4) provide
resonance in speech. The nasopharynx opens into the oropharynx
(behind the oral cavity). The oropharynx leads to the laryngopharynx,
and empties into the larynx (voice box), which contains the vocal cords,
passing through the glottis, connecting to the trachea (wind pipe).

30
Lower respiratory tract/respiratory zone
The trachea leads down to the thoracic cavity (chest) where it divides
into the right and left "main stem" bronchi. The subdivision of the
bronchus is: primary, secondary, and tertiary divisions (first, second
and third levels). In all, they divide 16 more times into even smaller
bronchioles.

The bronchioles lead to the respiratory zone of the lungs which consists
of respiratory bronchioles, alveolar ducts and the alveoli, the multi-
lobulated sacs in which most of the gas exchange occurs.

Upper respiratory infections

Commonly referred to the acronym URI, is the illness caused by an
acute infection which involves the upper respiratory tract: nose,
sinuses, pharynx, larynx, or bronchi. In the United States, this
represents approximately one billion acute upper respiratory illnesses
annually.

31
AIRBORNE TRANSMISSION
Many microbial pathogens have an airborne mode of transmission and
cause infections of the upper respiratory tract (URT). The infections
caused by such airborne organisms tend to occur in epidemic form,
which is the most infective form in nature.

Definition -Epidemic Form: Appearing explosively in nature and

attacking large no. of people within a short time. Their incidence
usually increases during the fall and winter when people are more
likely to occupy crowded quarters. The causative microorganisms
occur in secretions from the nose and throat of infected individuals and
they can be transmitted directly to healthy individuals by aerosols.

AEROSOLS:
Aerosols are fine sprays, producing droplets that remain suspended in
air for a time, are generated by coughs, sneezes or even talking.
Biological contaminants suspended in air are referred as aerosols.

Microorganisms that cause respiratory infections can also be

transmitted indirectly via fomites such as drinking glasses, eating
utensils and handkerchiefs that have recently been used by an infected
person. Also spreads as a droplet and droplet nuclei.

Infectious dusts –
Nosocomial diseases are caused by this dust. Nosocomial diseases are
diseases acquired in a hospital. Here large aerosol droplets come
through nasal and throat discharges from the patients. Sweeping a floor
in the patient’s room can generate dust particles which add
microorganisms to the circulating air which may cause significant
hazards to others.

Some examples of airborne diseases:

1. Tubercule bacilli – isolated from the dust of Sanitoria.

32
 2. Diptheria bacilli – floor dust near patients or carriers harboring
these.
3. Hemolytic streptococci – also from floor dust near patients or
carriers harboring these.

BACTERIAL INFECTIONS:
1. Diphtheria

History: (based on Greek dipthera “pair of leather scrolls”), is an upper

respiratory tract illness characterized by sore throat, low-grade fever,
and an adherent membrane (a pseudomembrane) on the tonsil(s),
pharynx, and/or nose. A milder form of diphtheria can be restricted to
the skin. It is caused by Corynebacterium diphtheriae, a facultatively
anaerobic Gram-positive bacterium. Diphtheria was named in 1826 by
French physician Pierre Bretonneau. The name alludes to the leathery,
sheath-like membrane that grows on the tonsils, throat, and in the nose.
The pronunciation ‘dip’ was originally considered incorrect, but has
become the most common way of saying the word, and is accepted as a
correct form. While many writers today use the spelling "diptheria"
which fits the modern pronunciation, this spelling is rarely found in
dictionaries. Diptheria derives its name from the whitish gray veil or
membrane that forms on the tonsils and pharynx.

Source: Diphtheria is a highly contagious disease spread by direct

physical contact or breathing the aerosolized secretions of infected
individuals.

Causative Agent: Cornebacterium diptheriae colonises the

nasopharynx where it forms a whitish gray membrane over the tonsils
and nasopharynx and produces the toxin that is carried by the
circulatory system to all parts of the body.

Symptoms: Slight fever, fatigue, malaise and a sore throat that is often
accompanied by dramatic swelling of the neck. An untreated patient’s
health can deteriorate rapidly and death from progressive organ failure
due to the toxin can follow.

33
Cornebacterium diptheriae is a gram positive straight or slightly curved
rod shaped bacterium.

Control Measures: Diptheria is of historic importance because this

once dreaded killer disease of children was the first bacterial disease to
be treated and controlled by immunological procedures. Once quite
common, diphtheria has largely been eradicated in developed nations
through wide-spread vaccination. Boosters of the vaccine are
recommended for adults since the benefits of the vaccine decrease with
age; they are particularly recommended for those traveling to areas
where the disease has not been eradicated.

2. Pertussis (Whooping Cough)

Causative Organism: Caused by Bordetella pertussis and Bordetella

parapertussis.

Symptoms: Mild cough, sneezing and inflammation of the nasal

mucous membrane.

Preventive Measure: DTP Vaccination.

Immunity: Recovery from whooping cough confers resistance to

subsequent infections but does not provide complete protection.
Immunization drive is necessary. The preferred drug for chemotherapy
is Erythromycin.

3. TB – Tuberculosis

There was a time, when TB caused one fourth of the all adult deaths in
all over the world. Despite modern methods of therapy, TB remains a
significant respiratory disease suffered annually by more than 20, 000
people. Early diagnosis and lengthy antimicrobial therapy are the
cornerstones of public health efforts to control tuberculosis.

Causative Agent: Mycobacterium tuberculosis or tubercle bacilli – It

resists destruction by phagocytosis and multiply intracellulary which is
the unique characteristics of tuberculosis.

34
Source: Droplet nuclei generated by coughing of an infected person.
It enters the alveoli upon being inhaled. A nonspecific inflammatory
reaction develops at the infection site and the tubercle bacilli are
engulfed by phagocytosis. Many bacilli are killed but some survive
and multiply as intracellular parasites. Infections spread by means of
these infected leukocytes to the regional to the regional lymph nodes
and from there to other parts of the body, where additional foci of
infection are established.

Montoux Test: It is the tuberculin test which is done by intradermally

injecting a purified protein derivative (PPD) taken from culture filtrates
of M.tuberculosis in a person, who tests positive, a red, hardened area
will appear at the site of infection in about 48 hours. A positive
reaction indicates that the person either has an active case of
tuberculosis or previously infected or has been immunized. A positive
chest film, the presence of acid-fast bacteria in sputum or biopsy
material and isolation and speciation of mycobacterium confirm the
diagnosis.

BCG Vaccine:

The Bacilli Calmette Guerin (BCG) is made with an attenuated strain of

mycobacterium and is administered to persons. The BCG
immunization is effective in preventing childhood tuberculosis through
its efficacy in preventing adult TB is still in question.

Infection & reactivation of dormant tubercule in previously infected

person. Infection is fresh spreads through droplet nuclei through
coughing of infected persons. Reactivation is the cause of 75% of the
cases. Crowded living conditions facilitate these active cases to others.

Drugs:

Combinations of isoniazid, rifampin and ethambutol are used to treat

TB.

ISONIAZID or simply INH, as it is known is widely used because it is

effective, is only slightly toxic and is inexpensive.

35
Important point to note: If a person has once been infected with
M.tuberculosis, (s) he will have to be continually vigilant against a
recurrence because the tubercle bacilli survive intracellulary in dormant
tubercles.

4. Bacterial Pneumonia:

Streptococcus pneumonia & Mycoplasma pneumoniae & several other

bacteria are capable of causing pneumonia – inhabitant of URT.

Source: Inspired air containing respiratory secretions.

Prevention: Pneumococcal polysaccharide vaccine.

5. Q-Fever: Coxiella burnebi – an obligate intracellular parasite

that is enzootic in cattle, sheep and goats.

Sources: Through saliva and nasal secretions. Aerosols infected

animals as droplets and droplet nuclei and after birth too.

Q stands for Query – because the nature of illness was not understood
at first.

Prevention: Cured by tetracyclines

VIRAL RESPIRATORY INFECTIONS

6. The common cold:

This is one of the most annoying among viral infections.

Coryza – an acute inflammation of the nasal mucous membranes

associated with a profuse discharge is the hallmark of the common
cold. Many viruses can infect the nasal membranes bringing on
mucous secretions, chills, headache, sneezing, sore throat and cough.
A head cold is hardly life-threatening but it does cause significant
discomfort and absenteeism from work and school.

Some of the viruses associated with the common cold:

36
 1. Rhinoviruses – more than100 antigenic types
2. Corona viruses
3. Parainfluenza viruses
4. Adenoviruses
5. Respiratory syncytial viruses
6. influenza viruses
7. other unknown presumed viruses

Target population: Children are the chief sources of infection and

direct contact with the infected person is the main mode of
transmission by aerosol and as a larger particle of respiratory
excretions suspended in air.

Symptoms: Sneezing, nasal discharge, a sore dry, scratchy throat,

headache, cough, malaise and chills. Initially the nasal discharge is
colourless & watery but later it becomes heavy and tan-coloured.

Drugs:

1. Phenylepherine & Ephedrine (to decrease swelling and allow

nasal secretions).
2. Aspirin – to decrease headache and muscle pains.

7. Influenza A or B or Flu – Serious viral infection of LRT

In winter months, they appear as respiratory illness among school-age

children than in adults and preschool children.

Incubation period: 18 to 72 hrs.

Source – By direct contact or aerosols.

Lasting immunity against the influenza virus cannot occur because it is

able to create multiple antigenic subtypes.

Drugs: Amantadine and rimantadine

8 Mumps: Swelling of the parotid glands

37
Causative agent: Mumps virus

Vaccine: Mumps vaccine – using live attenuated mumps virus MMR –

at fifteen months of age.

Systemic Mycosis: Begin as pulmonary lesions when the fungal

spores are inhaled in aerosols generated from soils that harbour natural
populations of the fungus.

9 Hospital Associated Infectious Diseases:

Hospital associated diseases are those that develop in the patient during
his stay in the hospital and that were not present at the time of
admission. Hospital associated diseases are called nosocomial, which
is derived from the Greek.

‘noses’ – disease

‘Komeion’ – to take care of.

A recent study of patient indicates that from 5% to 10% of the patients

in acute care hospitals will develop a nosocomial disease. Nosocomial
diseases are an important health problem, not only, because of the
increased risk on the patient’s life but also for economic reasons i.e.
approximately 54 million people are admitted to acute care hospitals
each year. If 5% acquired nosocomial diseases and require the average
7 days of additional care, the cost of the excess hospitalization is more.

Sources of Infection: The organism causing diseases in the hospital

may be from endogenous or exogenous sources.

Endogenous sources: are those from the patient’s own flora.

Exogenous sources: are those that arise from sources other than the
patient, which is made up of microorganisms that are considered
“nonvirulent” can cause endogenous diseases under circumstances.

38
 1. Te organism is displaced from its normal habitat (under such
circumstances, the new habitat often promotes the rapid
multiplication of the organism)
2. Conditions altering the environment of an organism, such as an
irritant (foreign object) or alteration of a blood supply to a site.
3. Multiple or broad spectrum antibiotics reduce the number of
competing microorganisms.

Exogenous sources of infection may be classified into animate &

animate

Animate Sources of infection include

1. Other patients in the hospital with infectious disease who may
represent a risk to other patients as well as hospital staff.
2. Hospital staff who serve as a reservoir of potentially infectious
microorganisms
3. Visitors who not only may serve as a source of infection but also
may themselves be at risk of acquiring an infection from the
patient

There are many inanimate objects within the hospital environment that
are sources of infection to the patient. The air may serve to transmit
any infectious agent that can survive outside the host for atleast a short
interval of time. Many fungal and bacterial spores are found naturally
in outside air and can be spread in the hospital by improperly designed
ventilating systems. Other microorganisms can be suspended in droplet
nuclei or dust particles and depending on air currents may be
transmitted for long distances. Respiratory agents such as those
causing TB are spread by coughing or sneezing. Staphylococci which
are found on the skin can be spread following desquamation (normal
peeling of the skin), particularly in patients with eczema and related
skin conditions. Patients with open wounds such as burn patients or
surgical patients are the most vulnerable to infection by the airborne
route.

39
Potential Hazards of Laboratory Techniques
Infection of laboratory workers is a common problem while handling
microorganisms. And in most of these cases finding out the source of
infection is very difficult.

The problem is very severe in clinical micro biology laboratories where

the microorganisms being handled are mostly pathogenic, although
other research and development laboratories experience a similar
problem.

The most common microorganisms causing laboratory acquired

infections are Brucella abortus, B. melitensis, Pasteurella tularensis and
Salmonella typhosa (Salle, 1974).

According to a survey conducted in 1978, the top ten laboratory

acquired infections include brucellosis, Q fever, hepatitis, typhoid
fever, tularaemia, tuberculosis, dermatomycosis, Venezulan equine
encephalitis, psittacosis and coccidioidomycosis. Of these tuberculosis
is becoming a serious hazard to laboratory workers especially in
mortuary and post mortem workers and in microbiology technicians.
Infection is mainly due to the inhalation of aerosols generated during
the work.

Three possible routes have been suggested to be responsible for

laboratory acquired infections (Fallon & Gnst, 1989). Inoculation may
be because of accidental introduction of infection into the body.
Examples of this type include introduction of infection into the eye by
splashing, into the body tissues through a needle injury, incision with a
sharp instrument or broken glass etc. Ingestion - infection through oral
route.

This includes accidental swallowing of infective material as in the case

of mouth pippeting of cultures. Inhalation of aerosols or dust articles
containing pathogenic microorganism is the third possible route.
Among the three it is the most hazardous one.

40
 Aerosols may be generated by any action that breaks the continuity of
the surface of a liquid, such as withdrawal of loop from broth culture.
These aerosol particles upon drying may become infectious dust.
Usually spilt culture fluid on a table or bench dries out and when
disturbed by actions like cleaning, becomes airborne infective dust.

41
 Laboratory-Acquired Viral Infections
The following laboratory-acquired viral infections had been reported;-

• Hepatitis A, B, and C - they account for the majority of known

laboratory-acquired infections
• Influenza, adeno, and mumps viruses
• Polio and coxsackieviruses
• Lassa fever (only two reported instances), Marburg, Crimean-
Congo, Yellow Fever, Dengue and Hantaviruses
• VEE, EEE, Rift Valley fever, Chikungunya, Kyasanur Forest
Disease, Japanese B encephalitis, West Nile, St Louis, Russian
spring-summer, and Louping ill and many other arboviruses
• HIV (two cases)
• Rabies (two reports)

Infections had occurred in widely different kinds of laboratories. Some

of the organisms are handled only in research establishments whilst
others are encountered daily in diagnostic and clinical laboratories. The
vast majority of reported infections occur in research institutes although
a wider population is at risk in a routine diagnostic laboratory.
Laboratory-acquired infections are far more likely to occur in untrained
workers. In laboratory-acquired infections, the route may not be the
same as the natural route. Routes of infection reported are;-

1. Oral - eating, drinking, and smoking in the laboratory, mouth

pipetting, transfer of microorganisms to mouth by contaminated
fingers or articles
2. Through the skin - injuries by needles, sharp instruments, or
glass. Animal bites and scratches. Cuts and scratches.
3. Through the conjunctiva - splashes of infectious material into
the eye, transfer of microorganisms to eyes by contaminated
fingers
4. Through the lungs - inhalation of airborne microorganisms

The mains sources of laboratory-acquired infections are accidents,

animals, clinical specimens, aerosols and work with the agent. The

42
types of accidents involved consist mainly of spillage and splashes,
needle and syringe, sharp objects and broken glass, animal scratch or
bites.

Air Sanitation - Sanitation of air is essential in enclosed places like

hospital wards, operation theatres and burns unit to prevent infection.
Food processing and packaging industries, pharmaceutical industries
and rooms where sterile materials or products are stored require aseptic
atmosphere to prevent contamination and to ensure safe handling.
Sterilization of large volumes of air has also become essential for aerobic
industrial fermentations. As a result of these, air sanitation has become an
important area in air-microbiology. Sanitation of air can be effected in a number of
ways each having its own applications.

1. Use of chemicals,

2. Mechanical methods,

3. Ultraviolet light,

4. Electrostatic precipitation and

5. Heating methods.

Air Sanitation By Chemical Agent - Air sanitation can be done by the

use of certain gaseous chemical agents. These agents are mostly used to
sterilize air in an enclosed space. Limitations with chemical agents are

(1) It is difficult to maintain a desired concentration because of the

deposition of the agents on surfaces and

(2) Large volumes of agents are required to maintain the final

concentration. Although no chemical agent has been found to be
successful, the following are of some use.

Hypochlorous acid: Hypochlorous acid or a hypochlorite like sodium

hypochlorite in a final concentration of 1:2 million can be used to
sanitize air. This concentration is sufficient to cause a reduction of
bacteria as well as viruses like influenza virus.

43
As with other gaseous agents, the effectiveness of hypochlorous acid or
hypochlorite against airborne microorganisms depends upon the
moisture content of air. Slightly increased relative humidity has a
profound action. For example, rapid killing of streptococci and
staphylococci occurs at a relative humidity of 90%.

Quaternary ammonium surface active disinfectant: This is a

commercially available disinfectant and can be used as an air sanitizing
agent. There occurs a reduction in the number of airborne and surface
bacteria in hospital rooms when this compound is spray-fogged.
Fogging procedures are effectively used to decontaminate the rooms
vacated by patients infected with staphylococci, streptococci,
pseudomonads and Salmonella.

Glycols: Propylene glycol and triethylene glycol are active against

streptococci, staphylococci, pneumococci, H. influenzae and influenza
virus at a concentration 1:4 million. Their microbicidal activity is
maximum at a temperature of about 27°C and a relative humidity of 45
- 70%.

The bactericidal activity of glycols is due to their hygroscopicity. When

glycol molecules are atomized into the air they dissolve in the film of
moisture surrounding each microorganism. At a particular
concentration of glycol, the moisture inside the bacterial cell is drawn
out of the cell and this leads to the death of the microbe.

Air Sanitation By Mechanical Methods -Mechanical methods are

aimed at the removal reduction of microorganisms.

Suppression of dust: Dust particles act as a substratum for

microorganisms to adhere Microorganisms can remain viable for quite
long period in these dust particles. Depending upon the factors such as
air current and weight of the particle, bacteria carrying dust particles
can either remain suspended in air or they may settle down on various
objects.

44
Thus dust particles play an important role in the dispersion of
microorganisms in air. So any procedure that suppresses the emergence
or distribution of dust will in turn affect the microflora of air.

Applying oil emulsion to floors, bed cloths and other textiles will
provide an effective control over dust and dust borne bacteria. Oil
mechanically inhibits the spread of dust by binding to them. Oiling
methods are simple in procedure and are practicable.

Even cost wise also, they are economical. Various studies have shown
that oiling floors and bed cloths in hospitals have considerably reduced
the incidence of respiratory tract infections. Removal of dust using
vacuum pump followed by application of disinfectant solution has also
been recommended.

Filters: Filtration can also be used as a method of air sanitation. Most

of the airborne microorganisms are present in dust particles of size
larger than 5/lm. Hence the microorganisms can be removed from air
by passage through simple filters, which can retain particles of this size.
If the smaller particles are also important then high efficiency filters
can be used.

The various types of filter materials used in air sterilization are 1.

Granular - activated charcoal; 2. Fibrous pads - cotton wool, slag wool,
and glass wool; and 3. Filter papers - cellulose - asbestos and glass
fibre.

HEPA Filters: High efficiency particulate air (HEPA) filters are

specially designed filters to deliver clean, sterile air into an enclosed
room or cabinet. The filter material is made up of fiberglass. HEPA
filters have an effective pore size of about 0.3/lm and an efficiency of
99.9%. Usually disposable pre-filter, which reduces the load to be
removed by the main filter, is used along the main filter.

The main use of HEPA filter is in laboratory safety cabinets where the
incoming air is filtered and the used air is decontaminated as the air
passes through the filter. HEPA filters are also used in hospitals to
provide sterile air. The limitation with HEPA filter is the cost.

45
Laminar air flow: Laminar air flow is again a kind of mechanical
method for air sanitation. Laminar air flow refers to the unidirectional
flow of air. Air is continuously flowing at high pressure in one
direction. When the air flows over a place, where microorganisms are
handled, the flow will carry all the escaping microorganisms to an
outlet.

Thus, the flow prevents the dissemination of microorganisms into

outside air. In safety cabinets laminar air flow is used to carry the used,
contaminated air away from the working person. To make the laminar,
air flow efficient HEPA filter can be used.

In this way sterile air can be produced and allowed to carryover the
used air. In industries, where hot materials to be cooled off aseptically,
laminar airflow tunnels in conjunction with HEPA filters can be used.

Air Sanitation by Ultraviolet Radiation - Radiation of short wave

lengths are more powerful in controlling airborne microorganisms.
Among these ultraviolet (UV) is the widely used one. Ultraviolet
radiation has a wavelength range of 210-328 nm. The maximum
microbicidal effect of UV light is considered to be around 260nm. This
wavelength is also the peak absorption wavelength for DNA.

Modem low pressure mercury vapour lamps emit more than 95% of
their radiation at 253.7nm and this is at the maximum microbicidal
activity. Thus about 50% of the total energy input to the lamp is
transmitted as effective UV radiation through the special glass in the
lamp. About 2% is transmitted into visible light. The remaining 48% is
transformed into heat.

Target sites and microbial inactivation mechanisms: The main

target site for UV radiation is the DNA. Various photo products
accumulate in UV exposed microbes. Exposure of non sporulating
bacteria to UV radiation results in the formation of purine and
pyrimidine dimers between adjacent molecules in the same strand of
DNA. UV also induces nucleic acid - protein cross links.

46
In Deinococcus radiodurans another type of photo product, 5, 6 -
dihydroxy dihydrothymine, has been found on exposure to UV
radiation. In bacterial spores yet another type of photo product, 5
thyminyl 5-6 dihydro thymine (TDHT), accumulates in DNA. Unless
removed, these photo products form non coding lesions in DNA which
ultimately lead to cell death.

Factors Affecting UV Sterilization - Several factors have been shown

to influence UV sterilization. Important factors among them are
discussed below.

Type of organism: Bacterial spores are found to be more resistant than

the vegetative cells. Comparatively, mold spores are highly resistant to
UV radiation. Viruses can also be inactivated by UV radiation and their
level of resistance is in between that of bacterial spores and non-
sporulating bacteria. Certain bacteria are highly resistant to UV
radiation by nature. Examples include Deinococcus radiodurans and
Sarcina lutea.

Type of suspension: When tested in the form of a dust suspension, B.

subtilis spores, show more resistance than when tested as an aerosol. In
case of most non sporulating bacteria, slightly increased dose of UV is
required for disinfection of dried forms than for the wet forms.

Cell number: An increase in cell number requires a corresponding

increase in the UV dose.

Effect of Temperature: Except Deinococcus radiodurans most

microorganisms are super sensitive to UV at low temperatures i.e. only
in frozen condition.

Effect of organic matter: Inactivation of microorganisms in the

presence of organic matter requires an increased dose of UV radiation.

Repair mechanism: Under certain circumstances most bacterial

species repair the damage caused by UV radiation. The repair process
may involve either light repair or dark repair. [For a detailed account on
the repair mechanisms the reader can refer to chapter 9 of Molecular

47
Biology by David Freifelder] Other minor factors include humidity of
the atmosphere, air motion, strength and length of rays and volume of
space.

Applications: UV radiation has wide applications as a sterilizing agent.

It is used in room sanitations especially in hospital wards. Since UV
radiation is irritating to the face and eyes, three different methods of
installing the lamps have been employed, (1) on the side wall or (2)
from the ceiling of a room to irradiate the air above the 7 feet level or
(3) on the side wall to irradiate the floor and air below the 30 inch
level.

Air warmed by the radiation rises up and moves upward to the ceiling.
This forces the cooler air down where it is warmed again and rises.
This circulation of air actually dilutes the contaminated air with the
disinfected air.

UV radiation is also finding use in operating room sanitation. UV

irradiation was used in the operating rooms for the first time by Hart in
1968 in USA. In surgery, the tissues exposed during operation have to
be protected from pusforming and fever producing microorganisms.
Although other aseptic techniques are employed in the operating room
it is better to use UV irradiation for air sanitation.

UV irradiation can be employed for product sanitation where aseptic

packing or processing is needed. Pharmaceutical industries, where
aseptic handling and packing of instruments are done, UV irradiation is
practiced.

Similarly, in food industries aseptic packing, processing and storing

require UV irradiation. By UV disinfection of enclosed air within the
domestic refrigerators, odors developing in stored foods can be
reduced. In addition it also provides some protection.

In cold store rooms meat is stored at 1.1-2.2°C. At this temperature

humidity drops down and as a result the meat dries out. When UV
lamps are used in these rooms, temperature can be increased to 7.2°C.
This allows higher humidities, thus reducing dehydration.

48
In ventilated safety cabinets and aseptic laboratory rooms, where
microorganisms are handled, UV irradiation is used for air sanitation.
In addition UV radiation has other uses like disinfection of drinking
water.

Disadvantages of UV Sterilization - When compared with other types

of radiations UV radiation is less effective because of its low
penetrating power. The quantum of energy liberated is low in case of
UV and this account for the low penetrating power.

Hence, it is not effective against the bacteria embedded in particles. It

requires costly equipment. It has low efficiency at low humidities. It
has certain harmful effects on eyes and skin.

Air Sanitation by Electrostatic Precipitation - In electrostatic

precipitation airborne dust particles containing microorganisms are
subjected to electric field. When the dust containing air is passed
through an ionizer the dust particles are become charged.

From the ionizer the charged particles are carried through a collector
which contains both negative and positive electrodes. Charged dust
particles are deposited on the electrodes of opposite charge as they pass
through them. Though it is a costly process, it is highly efficient

Air Sanitation by Heat Sterilization - Heating can also be used as an

air sanitation method. Sterile air can be produced by passage through a
heated pipe. The temperature of air must exceed 220°C to kill spores
and vegetative forms. Because of the complexity and expense this
method is employed very rarely.

Laminar Flow Hoods & Biological Safety Cabinets

What are HEPA Filters?

1. HEPA stands for High Efficiency Particulate Air
2. they consist of a thin pleated sheet of boron silicate microfibres
with aluminum separators

49
 3. they are particulate filters which retain airborne particles and
microorganisms (gases pass freely through)
4. filtration occurs by five distinct methods (*primary mechanisms):
• 1) Sedimentation
2) electrostatic attraction
3) interception
4) inertial impaction
5) diffusion
How are HEPA filters rated?
1. HEPA filters are rated on their ability to retain particles of 0.3
microns in size
2. tested with PAO (poly alpha olefin) which produces particles of
0.3 microns in size
3. >99.97% of these particles are retained by the HEPA filter
4. most aerosol droplets are greater than 0.3 microns
Do you know the difference between a laminar flow hood and a
biological safety cabinet?
Laminar Flow Hoods
1. provide product protection only and must not be used when
working with any form of biohazard or chemical hazard
2. any potentially infectious aerosol that is created will lead to
exposure of the operator and the environment
3. Horizontal-flow clean-air bench used for cell cultures can expose
the researcher to aerosols of allergenic or infectious materials.
4. vertical-flow clean-air bench also blows air out into the room

50
Biological Safety Cabinets
1. provide personnel and environmental protection and commonly
product protection
2. infectious agents must be used in a biological safety cabinet NOT
a laminar flow hood

Do you know the difference between Class I, II, & III Biological
Safety Cabinets?
Class I Biological Safety Cabinet:
1. a ventilated cabinet which provides personnel and environmental
protection only
2. air flow is directed away from the researcher, but is not HEPA
filtered, therefore there is no product protection
3. similar to a fume hood with a HEPA filter on the exhaust system
to protect against the release of biohazards
4. inward air flow ranges from 75-125 linear feet per minute (lfpm)
5. can be used with radioisotopes and some toxic chemicals
Class II Biological Safety Cabinet:
1. provides personnel, product and environmental protection
2. there are supply air and exhaust air HEPA filters
3. two general types: IIA cabinets have a minimum inward air flow
of 75 lfpm and recirculates 70% of the air; IIB cabinets have a
minimum inward air flow of 100 lfpm and exhaust either 70%
(type B1) or 100% (type B2)
4. most of the biological safety cabinets at UVic are Class II

51
Class III Biological Safety Cabinet:
1. these cabinets provide personnel, product and environmental
protection
2. they are hermetically sealed and all procedures are conducted
through arm-length rubber gloves
3. used in high level (Level 4) containment labs
4. there are two HEPA filters on the exhaust system
Are you sure you are using your Biological Safety Cabinet
correctly?
Procedures for Using Biological Safety Cabinets:
1. the cabinet must be turned on at least 5 minutes before starting
work in order to purge the air and remove any particulates
2. the researcher should wear a closed-front lab coat (or surgical
gown) and gloves
3. the gloves should overlap the cuffs
4. all materials needed for the manipulations should be placed in the
cabinet before the work is initiated to minimize in-and-out
motions
5. do not cover the air intake grill
6. the researcher should work well into the cabinet and not out close
to the front (at least four inches from the front grill)
7. when in use, the entry door to the lab (particularly in small
rooms) must be kept closed and traffic minimized
8. do not have electric fans blowing in the room when the biological
safety cabinet is in use; this will seriously effect the air flow of
the unit
9. develop procedures for the collection and decontamination of
waste materials to avoid clutter and minimize in-and-out motions

52
 10. the cabinet must be decontaminated with an appropriate
disinfectant at the end of each work operation
11. periodic use of 1-10% household bleach in water is
acceptable, but chlorine is corrosive (70% ethanol or quaternary
ammonium compounds may also be used if effective against
agent)
12. all biological safety cabinets must be certified for use when
first installed, any time the unit is moved or repaired, and on an
annual basis
13. all cabinets will have a certification sticker indicating the
last date of testing on the front face of the cabinet

Laminar flow (clean air) cabinets - these are not microbiological

safety cabinets. Air is drawn through HEPA filters and passed onto the
working surface and the room. They are widely used in pharmacies and
the preparation of tissue culture.

The siting of safety cabinets is important. The main problems are

caused b draughts from doors and windows and the movement of
people and therefore, safety cabinets should not be sired near doors.
HEPA filters are highly efficient in removing viable microorganisms
with a quoted efficiency rate is 99.997%. It is important to ensure that
ducted effluents are not discharged near to open windows, especially of
hospital wards

53
Class I Microbiological Safety Cabinet

Class II Microbiological Safety Cabinet

54
Class III Microbiological Safety Cabinet

Reference:

1. Microbiology by Anna K.Joshua

2. General Microbiology by Powar & Daginawala Vol. II Himalaya
Publishing House
3. General Microbiology by Boyd
4. Text Book of Microbiology by R.Anantha Narayanan &
C.K.Jayaram Paniker
5. www.wikepedia.org

55
 Chapter - 2

MICROBIOLOGY OF SEWAGE

When populations were small and separated, the amount of waste

materials available was also small and disposal of such small
quantities of wastes was not a problem. However, with an increase
in the population and with intensive agriculture and
industrialization, the amount of waste has steadily increased. This
has created serious problems of disposal.

It is estimated that an average American (for whom statistics are

available) uses about 3-4 lbs of food, 19 lbs of fossil fuels and about
150 gallons of water/day. This results in the production of about
120 gallon sewage, 4 lbs of trash and about 2 lbs of air pollutants.
The quantum of solid wastes available in other countries is
comparatively less. In India, it is estimated that the total solid
wastes available is about 109 million tons per annum.

Two major types of wastes recognized. They are

1. The solid wastes

2. The liquid wastes

In most Countries solid wastes are allowed to decay and decompose

in an open area away from the place of habitation. In some
countries these are buried in the ground and allowed to undergo
microbial degradation or used in landfillings. The liquid wastes
such as sewage are drained into the rivers or lakes or diluted into the
other irrigation waters after processing.

56
In large cities, domestic sewage is usually carried through a well-
planned sewer system and is collected at a place far away from the
place of habitation, processed and drained into rivers, lakes or sea.
With limiting amounts of freshwater available for both drinking as
well as for agricultural purposes, reclaiming a part of sewage has
become necessary.

It is now increasingly realized that for successful survival on this

planet, recycling of both organic and inorganic materials is essential.
Also the space for disposal of waste materials is becoming limiting.
Fortunately, there are a large number of microorganisms which can
degrade organic materials and purify the wastes to a point that they
can be reutilized. In the recycling of both solid and liquid wastes,
microbial activity is important. A variety of methods for waste
treatment are currently used but all of these depend on microbial
conversion or organic material into inorganic compounds
(mineralization).

Importance of Sewage Disposal: Waste water treatment is necessary

before waste water can be disposed of without producing significant
undesirable or even harmful effects. However, some communities
and municipalities still dispose of inadequately treated waste water
into natural bodies of water either because they are indifferent to the
consequences or because it is assumed that the body of water is
sufficiently large and so located that dilution prevents hazards.
Communities and municipalities can no longer rely on disposal of
waste water by dilution. There is an ever increasing demand for
domestic and industrial water, necessitating more reuse of waters
that receive waste waters. Disposal of inadequately treated waste
water leads to

1. Greater possibility for dissemination of pathogenic

microorganisms
2. Increased danger in using natural bodies of water for drinking
supplies
3. Contamination of oysters and other shellfish by pollution
making them unsafe for human consumption
4. Large losses in the waterfowl population chargeable to

57
 pollution of their winter feeding grounds
5. Increased danger of swimming in the water diminished value
of the water for the other recreational purposes
6. Depletion of the oxygen supply of the water by unstable
organic matter in sewage killing aquatic life.
7. Creation of miscellaneous objectionable conditions such as
offensive odours and accumulation of debris which decrease
property values
8. Accumulation and dissemination of toxic chemicals that
endanger ecosystems and threaten public health.

BOD

BIOCHEMICAL OXYGEN DEMAND

Definition: The quantity of oxygen required for the oxidation of

organic matter by bacterial action in the presence of oxygen. It is infact
a measure of the strength of organic matters in terms of its ability to
decrease oxygen in water. Mostly standard test contains in measuring
the oxygen depletion at 20˚C for five days. Tests can also be
performed at 25˚C and 30˚ C

It is an indicator of pollution in the aquatic environment. Because of a

high organic matter content sewage has a high BOD. It is the measure
of effective waste water treatment. The biochemical oxygen demand is
the requirement for molecular oxygen that accompanies the molecular
oxidation of biodegradable substances in waste by microorganisms.
The major purpose of sewage treatment is to reduce this BOD before it
is disposed off. The term BOD is used to designate the oxygen
consuming capacity of a liquid which is the measure of the level of
degradable organic matter present. The BOD is measured by
incubating a sample of the liquid in a sealed container and the amount
of oxygen consumed in a definite interval of time (20˚ c/days) is
measured. A high BOD represents a large amount of degradable
organic matter in the sample. Unpolluted natural waters have little or
no BOD. A decrease in the BOD during sewage treatment is a
reflection of the effectiveness of stabilization of the organic to
inorganic materials. The method used for sewage purification depends

58
on the BOD level the presence of toxic materials and the natural waters
into which the treated sewage is to be discharged.

Effluents with high BOD pollute the environment into which they are
discharged by indirectly depleting the oxygen available for plant and
animal life. Ecologically balanced streams, rivers and lakes can
become anaerobic when high BOD effluents are discharged into them.
Such habitats select for fermenting bacteria and those that grow by
anaerobic respiration. These same environments become unsuitable for
aerobic animal life which die or leave the habitat.

BOD Level (in

Water Quality
ppm)
1-2 Very Good-not much organic waste present
3-5 Moderately clean
6-9 Somewhat polluted
10+ Very polluted

Method

The BOD test is carried out by diluting the sample with de-ionized
water saturated with oxygen, inoculating it with a fixed aliquot of seed,
measuring the dissolved oxygen and sealing the sample (to prevent
further oxygen dissolving in). The sample is kept at 20 °C in the dark
(to prevent photosynthesis and thereby the addition of oxygen) for five
days and the dissolved oxygen is measured again. The difference
between the final D.O and initial D.O is the B.O.D. The apparent BOD
for the control is subtracted from the control result to provide the
corrected value.

The loss of dissolved oxygen in the sample, once corrections have been
made for the degree of dilution, is called the BOD5. In the UK
allylthiourea is also added at the start of the test to prevent oxidation of
ammonia. Results from such tests are represented as BOT5(ATU) and
referred to as Carbonaceous BOD (CBOD) in the U.S.. Less frequently
used is the Ultimate BOD (UBOD) test, in which DO is repeatedly

59
measured by DO meter in the same specialized bottles until it has
reached equilibrium.

BOD is similar in function to chemical oxygen demand (COD), in that

both measure the amount of organic compounds in water. However,
COD is less specific since it measures everything that can be
chemically oxidized rather than just levels of biologically active
organic matter.

BOD is used as a gauge of the effectiveness of wastewater treatment

plants. Various commercial devices are available for its determination.

BOD can be calculated by: Undiluted; Initial D.O - Final D.O = BOD
Diluted; ((Initial D.O - Final D.O)- BOD of Seed) X Dilution Factor

COD

Chemical Oxygen Demand:

The weight of oxygen taken up by the total amount of organic matter in

a sample of water without distinguishing between biodegradable and
nonbiodegradable organic matter. The result is expressed as the
number of parts per million (ppm) or mgms/litre or g/m3 of oxygen
taken up from a solution of boiling potassium dichromate in 2 hours.
The test has been used for assessing the strength of sewage and trade
wastes.

This is the satisfactory method for determining the organic load of a

water body, which is preferable to the Biochemical Oxygen Demand
(BOD). It is a rapidly measurable parameter for stream and industrial
waste studies and control of water treatment plants. The method is
based on the chemical oxidation of material in the presence of a
catalyst by Cr2O7 in 50% H2SO4.

3(CH2O) + 16 H + + 2 Cr2O7 2- Æ 4Cr3 + 3 CO2 + 11 H2O

The amount of unreacted Cr2O7 2- is then determined by titration with a

standard Mohr’s salt solution.

60
Ag2So4 catalyses the oxidation of straight chain aliphatic compounds,
aromatic hydrocarbons and pyridine. HgSo4 ties up Cl– as soluble
complex and prevent its interference.

SEWAGE OR WASTE WATER TREATMENT & DISPOSAL

Waste water treatment is necessary before wastewater can be disposed

of without producing significant undesirable or even harmful effects.
Some communities and municipalities still dispose off inadequately
treated wastewater into natural bodies of water, either because they are
indifferent to the consequences or because it is assumed that the body
of water is sufficiently large and so located that dilution prevents
hazards. Communities and municipalities can no longer rely on
disposal of waste water by dilution. There is an ever increasing
demand for domestic and industrial water, necessitating more reuse of
waters that receive wastewaters. Disposal of inadequately treated
waste water may lead to many problems.

Sewage treatment

Sewage treatment, or domestic wastewater treatment, is the process

of removing contaminants from wastewater, both runoff and domestic.
It includes physical, chemical and biological processes to remove
physical, chemical and biological contaminants. Its objective is to
produce a waste stream (or treated effluent) and a solid waste or sludge
also suitable for discharge or reuse back into the environment. This
material is often inadvertently contaminated with toxic organic and
inorganic compounds.

Sewage is created by residences, institutions, and commercial and

industrial establishments. It can be treated close to where it is created
(in septic tanks or on site package plants and other aerobic treatment
systems), or collected and transported via a network of pipes and pump
stations to a municipal treatment plant (see sewerage and pipes and
infrastructure). Sewage collection and treatment is typically subject to
local, state and federal regulations and standards (regulation and
controls). Industrial sources of wastewater often require specialized
treatment processes (see Industrial wastewater treatment).

61
Typically, sewage treatment involves three stages, called

1. Primary,
2. Secondary and
3. Tertiary treatment.

First, the solids are separated from the wastewater stream. Then
dissolved biological matter is progressively converted into a solid mass
by using indigenous, water-borne bacteria. Finally, the biological solids
are neutralized then disposed of or re-used, and the treated water may
be disinfected chemically or physically (for example by lagoons and
micro-filtration). The final effluent can be discharged into a stream,
river, bay, lagoon or wetland, or it can be used for the irrigation of a
golf course, green way or park. If it is sufficiently clean, it can also be
used for groundwater recharge.

Description

Raw influent (sewage) is the liquid waste from toilets, baths, showers,
kitchens, sinks etc. Household waste that is disposed of via sewers. In
many areas sewage also includes some liquid waste from industry and
commerce. In the United Kingdom, the waste from toilets is termed
foul waste, the waste from items such as basins, baths, kitchens is
termed sullage water, and the industrial and commercial waste is
termed trade waste.

The division of household water drains into greywater and blackwater

is becoming more common in the developed world, with greywater
being permitted to be used for watering plants or recycled for flushing
toilets. A lot of sewage also includes some surface water from roofs or
hard-standing areas. Municipal wastewater therefore includes
residential, commercial, and industrial liquid waste discharges, and
may include stormwater runoff. Sewage systems capable of handling
stormwater are known as combined systems. Such systems are usually

62
avoided since they complicate and thereby reduce the efficiency of
sewage treatment plants owing to their seasonality. The variability in
flows also leads to often larger than necessary, and subsequently more
expensive, treatment facilities. In addition, heavy storms that contribute
more flows than the treatment plant can handle may overwhelm the
sewage treatment system, causing a spill or overflow (called a
combined sewer overflow, or CSO, in the United States). It is
preferable to have a separate storm drain system for stormwater in areas
that are developed with sewer systems.

The construction of combined sewers is a less common practice in the

U.S. and Canada than in the past and is no longer accepted within
building regulations in the UK and other European countries. Instead,
liquid waste and stormwater are collected and conveyed in separate
sewer systems, referred to as sanitary sewers and storm sewers in the
U.S. and as foul sewers and surface water sewers in the UK.

As rainfall runs over the surface of roofs and the ground, it may pick up
various contaminants including soil particles and other sediment, heavy
metals, organic compounds, animal waste, and oil and grease. Some
jurisdictions require stormwater to receive some level of treatment
before being discharged directly into waterways. Examples of treatment
processes used for stormwater include sedimentation basins, wetlands,
buried concrete vaults with various kinds of filters, and vortex
separators (to remove coarse solids).

The site where the raw wastewater is processed before it is discharged

back to the environment is called a wastewater treatment plant
(WWTP). The order and types of mechanical, chemical and biological
systems that comprise the wastewater treatment plant are typically the
same for most developed countries:

• Mechanical treatment;

Influx (Influent)
Removal of large objects
Removal of sand and grit
Pre-precipitation

63
 • Biological treatment;

Oxidation bed (oxidizing bed) or aeration system

Post precipitation
Effluent

• Chemical treatment (this step is usually combined with settling

and other processes to remove solids, such as filtration. The
combination is referred to in the U.S. as physical-chemical
treatment.).

Treatment stages
Primary treatment

Primary treatment removes the materials that can be easily collected

from the raw wastewater and disposed of. The typical materials that are
removed during primary treatment include to fats, oils, and greases
(also referred to as FOG), sand, gravels and rocks (also referred to as
grit), larger settleable solids including human waste, and floating
materials. This step is done entirely with machinery, hence the name
mechanical treatment.

Influx (influent) and removal of large objects

In the mechanical treatment, the influx (influent) of sewage water is

strained to remove all large objects that are deposited in the sewer
system, such as rags, sticks, condoms, sanitary towels (sanitary
napkins) or tampons, cans, fruit, etc. This is most commonly done with
a manual or automated mechanically raked screen. This type of waste is
removed because it can damage or clog the equipment in the sewage
treatment plant.

Sand and grit removal

Primary treatment typically includes a sand or grit channel or chamber

where the velocity of the incoming wastewater is carefully controlled to
allow sand grit and stones to settle, while keeping the majority of the

64
suspended organic material in the water column. This equipment is
called a detritor or sand catcher. Sand grit and stones need to be
removed early in the process to avoid damage to pumps and other
equipment in the remaining treatment stages. Sometimes there is a sand
washer (grit classifier) followed by a conveyor that transports the sand
to a container for disposal. The contents from the sand catcher may be
fed into the incinerator in a sludge processing plant, but in many cases,
the sand and grit is sent to a landfill.

Primary sedimentation tank at a rural treatment plant

Sedimentation

Many plants have a sedimentation stage where the sewage is allowed to

pass slowly through large tanks, commonly called "primary clarifiers"
or "primary sedimentation tanks". The tanks are large enough that
faecal solids can settle and floating material such as grease and oils can
rise to the surface and be skimmed off. The main purpose of the
primary stage is to produce a generally homogeneous liquid capable of
being treated biologically and a sludge that can be separately treated or
processed. Primary settlement tanks are usually equipped with
mechanically driven scrapers that continually drive the collected sludge
towards a hopper in the base of the tank from where it can be pumped
to further sludge treatment stages.

65
Secondary treatment

Secondary treatment is designed to substantially degrade the

biological content of the sewage such as are derived from human waste,
food waste, soaps and detergent. The majority of municipal and
industrial plants treat the settled sewage liquor using aerobic biological
processes. For this to be effective, the biota requires both oxygen and a
substrate on which to live. There are number of ways in which this is
done. In all these methods, the bacteria and protozoa consume
biodegradable soluble organic contaminants (e.g. sugars, fats, organic
short-chain carbon molecules, etc.) and bind much of the less soluble
fractions into floc. Secondary treatment systems are classified as fixed
film or suspended growth. Fixed-film treatment process including
trickling filters and rotating biological contactors where the biomass
grows on media and the sewage passes over its surface. In suspended
growth systems—such as activated sludge—the biomass is well mixed
with the sewage and can be operated in a smaller space than fixed-film
systems that treat the same amount of water. However, fixed-film
systems are more able to cope with drastic changes in the amount of
biological material and can provide higher removal rates for organic
material and suspended solids than suspended growth systems.

Roughing filters are intended to treat particularly strong or variable

organic loads, typically industrial, to allow them to then be treated by
conventional secondary treatment processes. Characteristics include
typically tall, circular filters filled with open synthetic filter media to
which wastewater is applied at a relatively high rate. They are designed
to allow high hydraulic loading and a high flow-through of air. On
larger installations, air is forced through the media using blowers. The
resultant wastewater is usually within the normal range for
conventional treatment processes.

Activated sludge

Activated sludge plants use a variety of mechanisms and processes to

use dissolved oxygen to promote the growth of biological floc that
substantially removes organic material. It also traps particulate material

66
and can, under ideal conditions, convert ammonia to nitrite and nitrate
ultimately to nitrogen gas, (see also denitrification).

Fluidized bed reactors

The carbon adsorption following biological treatment was particularly

effective in reducing both the BOD and COD to low levels. A fluidized
bed reactor is a combination of the most common stirred tank packed
bed, continuous flow reactors. It is very important to chemical
engineering because of its excellent heat and mass transfer
characteristics. In a fluidized bed reactor, the substrate is passed
upward through the immobilized enzyme bed at a high velocity to lift
the particles. However the velocity must not be so high that the
enzymes are swept away from the reactor entirely. This causes high
mixing; this type of reactors is highly suitable for the exothermic
reactions. It is most often applied in immobilized enzyme catalysis.

Filter beds (oxidising beds)

Trickling filter bed using plastic media

In older plants and plants receiving more variable loads, trickling filter
beds are used where the settled sewage liquor is spread onto the surface
of a deep bed made up of coke (carbonised coal), limestone chips or
specially fabricated plastic media. Such media must have high surface
areas to support the biofilms that form. The liquor is distributed
through perforated rotating arms radiating from a central pivot. The
distributed liquor trickles through this bed and is collected in drains at
the base. These drains also provide a source of air which percolates up
through the bed, keeping it aerobic. Biological films of bacteria,

67
protozoa and fungi form on the media’s surfaces and eat or otherwise
reduce the organic content. This biofilm is grazed by insect larvae and
worms which help maintain an optimal thickness. Overloading of beds
increases the thickness of the film leading to clogging of the filter
media and ponding on the surface.

Biological aerated filters

Biological Aerated (or Anoxic) Filter (BAF) combines filtration with

biological carbon reduction, nitrification or denitrification. BAF usually
includes a reactor filled with a filter media. The media is either in
suspension or supported by a gravel layer at the foot of the filter. The
dual purpose of this media is to support highly active biomass that is
attached to it and to filter suspended solids. Carbon reduction and
ammonia conversion occurs in aerobic mode and sometime achieved in
a single reactor while nitrate conversion occurs in anoxic mode. BAF is
operated either in upflow or downflow configuration depending on
design specified by manufacturer.

Secondary Sedimentation tank at a rural treatment plant

Membrane biological reactors

Membrane biological reactors (MBR) combines activated sludge

treatment with a membrane liquid-solid separation process. The
membrane component utilizes low pressure microfiltration or ultra
filtration membranes and eliminates the need for clarifaction and
tertiary filtration. The membranes are typically immersed in the
aeration tank (however, some applications utilize a separate membrane

68
tank). One of the key benefits of a membrane bioreactor system is that
it effectively overcomes the limitations associated with poor settling of
sludge in conventional activated sludge (CAS) processes. The
technology permits bioreactor operation with considerably higher
mixed liquor suspended solids (MLSS) concentration than CAS
systems, which are limited by sludge settling. The process is typically
operated at MLSS in the range of 8,000–12,000 mg/L, while CAS is
operated in the range of 2,000–3,000 mg/L. The elevated biomass
concentration in the membrane bioreactor process allows for very
effective removal of both soluble and particulate biodegradable
materials at higher loading rates. Thus increased Sludge Retention
Times (SRTs)—usually exceeding 15 days—ensure complete
nitrification even under extreme cold weather operating conditions.

The cost of building and operating a MBR is usually higher than

conventional wastewater treatment, however, as the technology has
become increasingly popular and has gained wider acceptance
throughout the industry, the life-cycle costs have been steadily
decreasing. As well, in developed urban areas where the footprint of the
treatment plant is considered a limiting factor MBR facilities can be
considered a desirable option.

Secondary sedimentation

The final step in the secondary treatment stage is to settle out the
biological floc or filter material and produce sewage water containing
very low levels of organic material and suspended matter.

Rotating biological contactors

Rotating biological contactors (RBCs) are mechanical secondary

treatment systems, which are robust and capable of withstanding surges
in organic load. RBCs were first installed in Germany in 1960 and have
since been developed and refined into a reliable operating unit. The
rotating disks support the growth of bacteria and micro-organisms
present in the sewage, which breakdown and stabilise organic
pollutants. To be successful, micro-organisms need both oxygen to live
and food to grow. Oxygen is obtained from the atmosphere as the disks

69
rotate. As the micro-organisms grow, they build up on the media until
they are sloughed off due to shear forces provided by the rotating discs
in the sewage. Effluent from the RBC is then passed through final
clarifiers where the micro-organisms in suspension settle as sludge. The
sludge is withdrawn from the clarifier for further treatment.

Tertiary treatment

Tertiary treatment provides a final stage to raise the effluent quality

before it is discharged to the receiving environment (sea, river, lake,
ground, etc.). More than one tertiary treatment process may be used at
any treatment plant. If disinfection is practiced, it is always the final
process. It is also called "effluent polishing".

Filtration

Sand filtration removes much of the residual suspended matter.

Filtration over activated carbon removes residual toxins.

Lagooning

Lagooning provides settlement and further biological improvement

through storage in large man-made ponds or lagoons. These lagoons are
highly aerobic and colonization by native macrophytes, especially
reeds, is often encouraged. Small filter feeding invertebrates such as
Daphnia and species of Rotifera greatly assist in treatment by removing
fine particulates.

Constructed wetlands

Constructed wetlands include engineered reedbeds and a range of

similar methodologies, all of which provide a high degree of aerobic
biological improvement and can often be used instead of secondary
treatment for small communities, also see phytoremediation. One
example is a small reedbed used to clean the drainage from the
elephants' enclosure at Chester Zoo in England.

70
Waste removal

Wastewater may contain high levels of the nutrients nitrogen and

phosphorus. Excessive release to the environment can lead to a build up
of nutrients, called eutrophication, which can in turn encourage the
overgrowth of weeds, algae, and cyanobacteria (blue-green algae). This
may cause an algal bloom, a rapid growth in the population of algae.
The algae numbers are unsustainable and eventually most of them die.
The decomposition of the algae by bacteria uses up so much of oxygen
in the water that most or all of the animals die, which creates more
organic matter for the bacteria to decompose. In addition to causing
deoxygenation, some algal species produce toxins that contaminate
drinking water supplies. Different treatment processes are required to
remove nitrogen and phosphorus.

Nitrogen removal

The removal of nitrogen is effected through the biological oxidation of

nitrogen from ammonia (nitrification) to nitrate, followed by
denitrification, the reduction of nitrate to nitrogen gas. Nitrogen gas is
released to the atmosphere and thus removed from the water.

Nitrification itself is a two-step aerobic process, each step facilitated by

a different type of bacteria. The oxidation of ammonia (NH3) to nitrite
(NO2−) is most often facilitated by Nitrosomonas spp.
(nitroso=ammonium). Nitrite oxidation to nitrate (NO3−), though
traditionally believed to be facilitated by Nitrobacter spp.
(nitro=nitrite), is now known to be facilitated in the environment
almost exclusively by Nitrospira spp.

Denitrification requires anoxic conditions to encourage the appropriate

biological communities to form. It is facilitated by a wide diversity of
bacteria. Sand filters, lagooning and reed beds can all be used to reduce
nitrogen, but the activated sludge process (if designed well) can do the
job the most easily. Since denitrification is the reduction of nitrate to
dinitrogen gas, an electron donor is needed. This can be, depending on
the wastewater, organic matter (from faeces), sulfide, or an added

71
donor like methanol.

Sometimes the conversion of toxic ammonia to nitrate alone is referred

to as tertiary treatment.

Phosphorus removal

Phosphorus can be removed biologically in a process called enhanced

biological phosphorus removal. In this process, specific bacteria, called
polyphosphate accumulating organisms are selectively enriched and
accumulate large quantities of phosphorus within their cells (up to 20%
of their mass). When the biomass enriched in these bacteria is separated
from the treated water, these biosolids have a high fertilizer value.

Phosphorus removal can also be achieved by chemical precipitation,

usually with salts of iron (e.g. ferric chloride) or aluminum (e.g. alum).
The resulting chemical sludge is difficult to handle and the added
chemicals can be expensive. Despite this, chemical phosphorus
removal requires significantly smaller equipment footprint than
biological removal, is easier to operate and can be more reliable in
areas that have wastewater compositions that make biological
phosphorus removal difficult.

Disinfection

The purpose of disinfection in the treatment of wastewater is to

substantially reduce the number of microorganisms in the water to be
discharged back into the environment. The effectiveness of disinfection
depends on the quality of the water being treated (e.g., cloudiness, pH,
etc.), the type of disinfection being used, the disinfectant dosage
(concentration and time), and other environmental variables. Cloudy
water will be treated less successfully since solid matter can shield
organisms, especially from ultraviolet light or if contact times are low.
Generally, short contact times, low doses and high flows all militate
against effective disinfection. Common methods of disinfection include
ozone, chlorine, or ultraviolet light. Chloramine, which is used for
drinking water, is not used in wastewater treatment because of its
persistence.

72
Chlorination remains the most common form of wastewater
disinfection in North America due to its low cost and long-term history
of effectiveness. One disadvantage is that chlorination of residual
organic material can generate chlorinated-organic compounds that may
be carcinogenic or harmful to the environment. Residual chlorine or
chloramines may also be capable of chlorinating organic material in the
natural aquatic environment. Further, because residual chlorine is toxic
to aquatic species, the treated effluent must also be chemically
dechlorinated, adding to the complexity and cost of treatment.
Ultraviolet (UV) light can be used instead of chlorine, iodine, or other
chemicals. Because no chemicals are used, the treated water's taste is
more natural and pure as compared to other methods. UV radiation
causes damage to the genetic structure of bacteria, viruses, and other
pathogens, making them incapable of reproduction. The key
disadvantages of UV disinfection are the need for frequent lamp
maintenance and replacement and the need for a highly treated effluent
to ensure that the target microorganisms are not shielded from the UV
radiation (i.e., any solids present in the treated effluent may protect
microorganisms from the UV light). In the United Kingdom, light is
becoming the most common means of disinfection because of the
concerns about the impacts of chlorine in chlorinating residual organics
in the wastewater and in chlorinating organics in the receiving water.
Edmonton, Alberta, Canada also uses UV light for its water treatment.
Ozone O3 is generated by passing oxygen O2 through a high voltage
potential resulting in a third oxygen atom becoming attached and
forming O3. Ozone is very unstable and reactive and oxidizes most
organic material it comes in contact with, thereby destroying many
pathogenic microorganisms. Ozone is considered to be safer than
chlorine because, unlike chlorine which has to be stored on site (highly
poisonous in the event of an accidental release), ozone is generated
onsite as needed. Ozonation also produces fewer disinfection by-
products than chlorination. A disadvantage of ozone disinfection is the
high cost of the ozone generation equipment and the requirements for
highly skilled operators.

73
Package plants and batch reactors

In order to use less space, treat difficult waste, deal with intermittent
flow or achieve higher environmental standards, a number of designs of
hybrid treatment plants have been produced. Such plants often combine
all or at least two stages of the three main treatment stages into one
combined stage. In the UK, where a large number of sewage treatment
plants serve small populations, package plants are a viable alternative
to building discrete structures for each process stage.

The most advanced packaged treatment plant according to a study done

by the University of California at Davis for the California State Water
Resources Control Board for treating waste and nutrients (phosphorus
and nitrogen) in one step economically is the USBFTM (Upflow Sludge
Blanket Filter). The USBFTM process is a modification of the
conventional activated sludge process that incorporates an anoxic
selector zone and an upflow sludge blanket filtration clarifier all in one
integrated bioreactor vessel. The treatment includes efficient reduction
of BOD5 and TSS but also biological nutrient removal (BNR) by the
processes of denitrification and "biological luxury uptake". The ensuing
compact, modular system takes up less space and contains very few
moving parts. The result is an efficient, highly affordable wastewater
treatment plant with low maintenance and operating costs.

USBFTM technology has no inherent capacity limits and is used in a

wide range of applications from subdivisions (Bay Point, FL) resorts
(Sun Peaks, BC, Kicking Horse, BC) and municipalities (Pahrump,
NV), to agricultural and industrial sites. Plants can be retrofitted and
expanded from existing sites reducing capital costs. Since there are no
mechanical parts and no chemicals needed, operations cost are much
less than sequencing batch reactor and membrane bio reactor systems.

Another type of process which combines secondary treatment and

settlement is the sequencing batch reactor (SBR). Typically, activated
sludge is mixed with raw incoming sewage and mixed and aerated. The
resultant mixture is then allowed to settle producing a high quality
effluent. The settled sludge is run off and re-aerated before a proportion

74
is returned to the head of the works. SBR plants are now being
deployed in many parts of the world including North Liberty, Iowa, and
Llanasa, North Wales.

The disadvantage of such processes is that precise control of timing,

mixing and aeration is required. This precision is usually achieved by
computer controls linked to many sensors in the plant. Such a complex,
fragile system is unsuited to places where such controls may be
unreliable, or poorly maintained, or where the power supply may be
intermittent.

Package plants may be referred to as high charged or low charged. This

refers to the way the biological load is processed. In high charged
systems, the biological stage is presented with a high organic load and
the combined floc and organic material is then oxygenated for a few
hours before being charged again with a new load. In the low charged
system the biological stage contains a low organic load and is
combined with floculate for a relatively long time.

Sludge treatment and disposal

The sludges accumulated in a wastewater treatment process must be

treated and disposed of in a safe and effective manner. The purpose of
digestion is to reduce the amount of organic matter and the number of
disease-causing microorganisms present in the solids. The most
common treatment options include anaerobic digestion, aerobic
digestion, and composting.

The choice of a wastewater solid treatment method depends on the

amount of solids generated and other site-specific conditions. However,
in general, composting is most often applied to smaller-scale
applications followed by aerobic digestion and then lastly anaerobic
digestion for the larger-scale municipal applications.

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Anaerobic digestion

Anaerobic digestion is a bacterial process that is carried out in the

absence of oxygen. The process can either be thermophilic digestion, in
which sludge is fermented in tanks at a temperature of 55°C, or
mesophilic, at a temperature of around 36°C. Though allowing shorter
retention time (and thus smaller tanks), thermophilic digestion is more
expensive in terms of energy consumption for heating the sludge.

One major feature of anaerobic digestion is the production of biogas,

which can be used in generators for electricity production and/or in
boilers for heating purposes.

Aerobic digestion

Aerobic digestion is a bacterial process occurring in the presence of

oxygen. Under aerobic conditions, bacteria rapidly consume organic
matter and convert it into carbon dioxide. The operating costs are
characteristically much greater than for anaerobic digestion because of
the energy costs needed to add oxygen to the process.

Composting

Composting is also an aerobic process that involves mixing the

wastewater solids with sources of carbon such as sawdust, straw or
wood chips. In the presence of oxygen, bacteria digest both the
wastewater solids and the added carbon source and, in doing so,
produce a large amount of heat.

Thermal depolymerization

Thermal depolymerization uses hydrous pyrolysis to convert reduced

complex organics to oil.

76
Sludge disposal

When a liquid sludge is produced, further treatment may be required to

make it suitable for final disposal. Typically, sludges are thickened
(dewatered) to reduce the volumes transported off-site for disposal.
There is no process which completely eliminates the need to dispose of
biosolids. There is, however, an additional step some cities are taking
to superheat the wastewater sludge and convert it into small pelletized
granules that are high in nitrogen and other organic materials. This
product is then sold to local farmers and turf farms as a soil amendment
or fertilizer, reducing the amount of space required to dispose of sludge
in landfills.

Treatment in the receiving environment

The outlet of a wastewater treating plant flows into a small river

Many processes in a wastewater treatment plant are designed to mimic

the natural treatment processes that occur in the environment, whether
that environment is a natural water body or the ground. If not
overloaded, bacteria in the environment will consume organic
contaminants, although this will reduce the levels of oxygen in the
water and may significantly change the overall ecology of the receiving
water. Native bacterial populations feed on the organic contaminants,
and the numbers of disease-causing microorganisms are reduced by
natural environmental conditions such as predation exposure to
ultraviolet radiation, for example. Consequently, in cases where the
receiving environment provides a high level of dilution, a high degree
of wastewater treatment may not be required. However, recent evidence
has demonstrated that very low levels of certain contaminants in
wastewater, including hormones (from animal husbandry and residue
from human birth control pills) and synthetic materials such as

77
phthalates that mimic hormones in their action, can have an
unpredictable adverse impact on the natural biota and potentially on
humans if the water is re-used for drinking water. In the US and EU,
uncontrolled discharges of wastewater to the environment are not
permitted under law, and strict water quality requirements are to be
met. A significant threat in the coming decades will be the increasing
uncontrolled discharges of wastewater within rapidly developing
countries.

Sewage treatment in developing countries

There are few reliable figures on the share of the wastewater collected
in sewers that is being treated in the world. In many developing
countries the bulk of domestic and industrial wastewater is discharged
without any treatment or after primary treatment only. In Latin America
about 15% of collected wastewater passes through treatment plants
(with varying levels of actual treatment). In Venezuela, a below
average country in South America with respect to wastewater
treatment, 97 percent of the country’s sewage is discharged raw into the
environment. Even a highly industrialized country such as the People's
Republic of China discharges about 55 percent of all sewage without
treatment of any type. In a relatively developed Middle Eastern country
such as Iran, Tehran's majority of population has totally untreated
sewage injected to the city’s groundwater. Most of sub-Saharan Africa
is without wastewater treatment.

Water utilities in developing countries are chronically underfunded

because of low water tariffs, the inexistence of sanitation tariffs in
many cases, low billing efficiency (i.e. many users that are billed do not
pay) and poor operational efficiency (i.e. there are overly high levels of
staff, there are high physical losses, and many users have illegal
connections and are thus not being billed). In addition, wastewater
treatment typically is the process within the utility that receives the
least attention, partly because enforcement of environmental standards
is poor. As a result of all these factors, operation and maintenance of
many wastewater treatment plants is poor. This is evidenced by the
frequent breakdown of equipment, shutdown of electrically operated
equipment due to power outages or to reduce costs, and sedimentation

78
due to lack of sludge removal. Developing countries as diverse as
Egypt, Algeria, China or Colombia have invested substantial sums in
wastewater treatment without achieving a significant impact in terms of
environmental improvement. Even if wastewater treatment plants are
properly operating, it can be argued that the environmental impact is
limited in cases where the assimilative capacity of the receiving waters
(ocean with strong currents or large rivers) is high, as it is often the
case.

Benefits of wastewater treatment compared to benefits of sewage

collection in developing countries

Waterborne diseases that are prevalent in developing countries, such as

diarrhea, typhus and cholera, are caused primarily by poor hygiene
practices and the absence of improved household sanitation facilities.
The public health impact of the discharge of untreated wastewater is
comparatively much lower. Hygiene promotion, on-site sanitation and
low-cost sanitation thus are likely to have a much greater impact on
public health than wastewater treatment. Given the scarcity of financial
resources in developing countries and the poor track record of
wastewater treatment plants, it could thus be argued that investments
should first be undertaken to evacuate wastewater from human
settlements and to promote good hygiene practices. Only once this has
been achieved, substantial funds should be invested in wastewater
treatment plants.

Contaminated drinking water contributes to disease in developing

and developed countries worldwide

Drinking-water quality is an issue of concern for human health in

developing and developed countries world-wide. The risks arise from
infectious agents, toxic chemicals and radiological hazards.
Experience highlights the value of preventive management
approaches spanning from water resource to consumer.

WHO produces international norms on water quality and human health

in the form of guidelines that are used as the basis for regulation and
standard setting, in developing and developed countries world-wide.

79
Drinking water is water that is intended to be ingested by humans.
Water of sufficient quality to serve as drinking water is termed potable
water whether it is used as such or not. Although many sources are
utilized by humans, some contain disease vectors or pathogens and
cause long-term health problems if they do not meet certain water
quality guidelines. Water that is not harmful for human beings is
sometimes called safe water, water which is not contaminated to the
extent of being unhealthy. The available supply of drinking water is an
important criterion of carrying capacity, the population level that can
be supported by planet Earth.

As of the year 2006 (and pre-existing for at least three decades), there
is a substantial shortfall in availability of potable water in lesser
developed countries, primarily arising from overpopulation. As of the
year 2000, 37 percent of the populations of lesser developed countries
did not have access to safe drinking water. Implications for disease
propagation are significant. Many nations have water quality
regulations for water sold as drinking water, although these are often
not strictly enforced outside of the developed world. The World Health
Organization sets international standards for drinking water. A broad
classification of drinking water safety worldwide can be found in Safe
Water for International Travelers.

The standard test for bacterial contamination is a laboratory analysis of

coliform bacteria, a convenient marker for a class of harmful fecal
pathogens. The presence of fecal coliforms (like Escherichia coli)
serves as an indication of contamination by sewage

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 Chapter - 3
AQUATIC MICROBIOLOGY
or
MICROBIOLOGY OF WATER
Introduction:

The quality of water is of vital concern for mankind since it is directly

linked with human welfare. It is known that faecal pollution of
drinking water caused water borne diseases. Generally speaking water
pollution is a state of deviation from the pure condition whereby its
normal function and properties are affected. Aquatic or water
microbiology is the study of microorganisms and their activities in
fresh, estuarine and marine waters. It is the study of microorganisms
like viruses, bacteria, algae, protozoa and microscopic fungi which
inhabit these natural waters.

Transcient microorganisms: Microorganisms entering the water from

air or soil or industrial or domestic wastes are called transcient
microorganisms.

Waste water usually contains microorganisms which will influence the

activities of microorganisms already present in the receiving waters.
Aquatic microorganisms and their activities are of great importance in
many ways. These may affect the health of human and other animal
life. They occupy a key position in the food-chain by providing rich
nourishment for the next higher level of aquatic life. They are
instrumental in the chain of biochemical reactions which accomplish
recycling of elements e.g. in mineralization.

81
Need to Preserve Water Resources:

Aquatic microbiology has emerged as one of the more important areas

of applied microbiology because of the following necessities.

1. Urbanization and consequently the growing demand for water by

communities.
2. The importance of natural water as major food source
3. The offshore exploration for oil and minerals and other
development

All these have resulted in the establishment of Federal Agencies which

exercise jurisdiction over many aspects of natural bodies of water.

1. The Environmental Protection Agency (EPA)

2. The National Oceanic & Atmospheric Administration (NOAA)

Kinds Of Water:

1. Atmospheric water
2. Surface Water
3. Ground Water

The earth’s moisture is in continuous circulation, a process known as

the water cycle or hydrologic cycle. Microorganisms of various kinds
are present at different stages of this cyclic process. Because the kinds
of aquatic environments are so different it is not surprising that
different species of microbes are considered to be indigenous to
specific habitats.

1. Atmospheric Water: The moisture contained in clouds and

precipitated as snow, slit, hail and rain constitutes atmospheric
water. The microbial flora of this water is contributed by the air.
In effect the air is ‘washed’ by atmospheric water which carries
with it the particles of dust to which microorganisms are
attached. Most of the microorganisms are thus removed from air
during the early stages of precipitation.
2. Surface Water: Bodies of water such as lakes, streams and rivers
and oceans represent surface water. To a greater or lesser degree,

82
 these waters are susceptible to contamination with
microorganisms from atmospheric water (precipitation), the
surface run off from soil and any wastes deliberately dumped into
them. Microbial populations vary in both number and kind with
the source of water, with composition of water in terms of
microbial nutrients and with geographical, biological and
climatic.
3. Ground Water: Ground water is subterranean water that occurs
where all pores in the soil or rock containing materials are
saturated. Bacteria as well as suspended particles are moved by
filtration in varying degrees depending on the permeability
characteristics of the soil and the depth to which water penetrates.
Springs consists of ground-water that reaches the surface through
a rock fissure or exposed porous soil. Wells are made by sinking
a shaft into the ground to penetrate the ground water level. Wells
less than 100 ft. deep are considered to be shallow.
Bacteriologically speaking wells and springs that are properly
located produce water of very good quality. If precautions are
taken to avoid contamination, the microbial content is negligible.

An aquatic ecosystem is an ecosystem located in a body of water.

Communities of organisms that are dependent on each other and on
their environment live in aquatic ecosystems. The two main types of
aquatic ecosystems are marine ecosystems and freshwater
ecosystems

Hydrologic Cycle:

It is important to know about hydrologic cycle or water cycle. Water

continuously circulates from the oceans to the atmosphere to the land
and back to the oceans providing us with a renewable supply of
purified water on land. This complex cycle known as the hydrologic
cycle results in a balance among water in the oceans, water on the land
and water in the atmosphere. When water evaporates from the Ocean’s
surface, it forms water bearing clouds in the atmosphere. Water also
evaporates from soil, streams, rivers and lakes.

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TRANSPIRATION:

Transpiration is the loss of water vapour from land plants also adds
water to the atmosphere. Roughly 97% of the water absorbed from the
soil by a plant is transported to the leaves where it is transpired back to
the environment.

84
Types of aquatic ecosystems

Aquatic ecosystems can be divided into two general types: marine

ecosystems and freshwater ecosystems.

Neritic (the relatively shallow part of the ocean that lies over the
continental shelf); profundal (bottom or deep water); benthic (bottom
substrates); intertidal (the area between high and low tides); estuaries

Marine ecosystems

A coral reef near the Hawaiian islands is an example of a complex

marine ecosystem.

Marine ecosystems cover approximately 71% of the Earth's surface and

contain approximately 97% of the planet's water. They generate 32% of
the world's net primary production. They are distinguished from
freshwater ecosystems by the presence of dissolved compounds,
especially salts, in the water. Approximately 85% of the dissolved
materials in seawater are sodium and chlorine. Seawater has an average
salinity of 35 parts per thousand (ppt) of water. Actual salinity varies
among different marine ecosystems.

Marine ecosystems can be divided into the following zones:

oceanicsh|salt marshes; coral reefs; and hydrothermal vents (where
chemosynthetic sulphur bacteria form the food base).

85
Classes of organisms found in marine ecosystems include brown algae,
dinoflagellates, corals, cephalopods, echinoderms, and sharks. Fish
caught in marine ecosystems are the biggest source of commercial
foods obtained from wild populations.

Environmental problems concerning marine ecosystems include

unsustainable exploitation of marine resources (for example overfishing
of certain species), water pollution, and building on coastal areas

An estuary is a semi-enclosed coastal body of water with one or more

rivers or streams flowing into it, and with a free connection to the open
sea. Estuaries are often associated with high rates of biological
productivity. An estuary is typically the tidal mouth of a river (aestus
is Latin for tide), and estuaries are often characterized by sedimentation
or silt carried in from terrestrial runoff and, frequently, from offshore.
They are made up of brackish water. Estuaries are more likely to occur
on submerged coasts, where the sea level has risen in relation to the
land; this process floods valleys to form rias and fjords. These can
become estuaries if there is a stream or river flowing into them. Large
estuaries, like Chesapeake Bay and Puget Sound often have many
streams flowing into them and can have complex shapes. Estuaries are
often given names like bay, sound, fjord, etc. The terms are not
mutually exclusive. Where an enormous volume of river water enters
the sea (as, for example, from the Amazon into the South Atlantic) its
estuary could be considered to extend well beyond the coast.

Freshwater ecosystems

Freshwater ecosystems cover 0.8% of the Earth's surface and contain

0.009% of its total water. They generate nearly 3% of its net primary
production. Freshwater ecosystems contain 41% of the world's known
fish species. There are three basic types of freshwater ecosystems:

• Lentic: slow-moving water, including pools, ponds, and lakes.

• Lotic: rapidly-moving water, for example streams and rivers.
• Wetlands: areas where the soil is saturated or inundated for at
least part of the time.

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Lake ecosystems can be divided into zones: pelagic (open offshore
waters); profundal; littoral (nearshore shallow waters); and riparian (the
area of land bordering a body of water). Two important subclasses of
lakes are ponds, which typically are small lakes that intergrade with
wetlands, and water reservoirs. Many lakes, or bays within them,
gradually become enriched by nutrients and fill in with organic
sediments, a process called eutrophication. Eutrophication is
accelerated by human activity within the water catchment area of the
lake

Lake ecosystems can be divided into zones: pelagic (open offshore

waters); profundal; littoral (nearshore shallow waters); and riparian (the
area of land bordering a body of water). Two important subclasses of
lakes are ponds, which typically are small lakes that intergrade with
wetlands, and water reservoirs. Many lakes, or bays within them,
gradually become enriched by nutrients and fill in with organic
sediments, a process called eutrophication. Eutrophication is
accelerated by human activity within the water catchment area of the
lake.

The major zones in river ecosystems are determined by the river bed's
gradient or by the velocity of the current. Faster moving turbulent water
typically contains greater concentrations of dissolved oxygen, which
supports greater biodiversity than the slow moving water of pools.
These distinctions form the basis for the division of rivers into upland
and lowland rivers. The food base of streams within riparian forests is
mostly derived from the trees, but wider streams and those that lack a
canopy derive the majority of their food base from algae. Anadromous
fish are also an important source of nutrients. Environmental threats to
rivers include loss of water, dams, chemical pollution and introduced
species.

Wetlands are dominated by vascular plants that have adapted to

saturated soil. Wetlands are the most productive natural ecosystems
because of the proximity of water and soil. Due to their productivity,
wetlands are often converted into dry land with dikes and drains and
used for agricultural purposes. Their closeness to lakes and rivers
means that they are often developed for human settlement

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Pond Ecosystems

This is a specific type of freshwater ecosystem that is largely based on

the autotroph algae which provide the base trophic level for all life in
the area. The largest predator in a pond ecosystem will normally be a
fish and in-between range smaller insects and microorganisms. It may
have a scale of organisms from small bacteria to big creatures like
water snakes, beetles, water bugs, and turtles

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 DRINKING WATER STANDARDS

Potable water is water suitable for drinking. It is estimated that man

can survive without food for 20 days but starts to struggle for life in the
absence of water just after one day. Water is needed for the
maintenance of life of plants and animals, for navigation, hydro-electric
power and for disposal of sewage. The most important contributors to
pollution of water are
1. sewage
2. oil and
3. industrial and agricultural wastes
These can be divided into
1. degradable and
2. non-biodegradable
Degradable pollutants, mostly domestic, sewage can be rapidly
decomposed by natural process. Non-degradable pollutants such as
inorganic chemicals like salts, chlorides, metallic oxides and toxic and
other waste producing materials are those substances in which there are
no evolved natural treatment produced that can keep up with the rate of
man made input eco-system. These either do not degrade or degrade
only very slowly in the natural environment. Water pollution not only
changes the physical properties of water such as colour, odour,
turbidity, taste and temperature but also makes it acidic, alkaline or
saline due to the presence of dissolved or suspended chemical
substances. Water pollution is caused due to physical, chemical and
biological impurities in water.

Physical Impurities:
Include turbidity, taste, colour and odour
1. Turbidity is caused by suspended and colloidal matter
2. Colour is due to the presence of mineralogical compounds
such as iron-oxide
3. Taste and odour are due to the presence in water of organic
matter dissolved during passage through the ground or from
industrial work, microorganisms such as algal growth.

89
Chemical Impurities:
Chemical impurities are due to carbonates and bicarbonates of
calcium and magnesium, sulphates and chlorides of calcium and
magnesium and carbonates-bicarbonates of sodium. Nitrates, chlorides
and fluorides of sodium, iron-oxide and manganese. They will create
turbidity, hardness and alkalinity, bad taste and odour problems.

Bacteriological Impurities:
Due to pathogenic bacteria bacteriological impurities arise in water.
Their presence is noted if E.coli bacteria are present. Bacteriological
tests involve the following:
1. Standard plate count
2. E.coli test

90
 QUALITY OF WATER FOR DRINKING

Supplies should be drawn from the best available source. If the source
cannot be adequately protected against pollution of water it must be
treated to ensure its safety. Possible hazards should be identified by
sanitary surveys and eliminated.

1. Bacteriological Quality:

The coliform index is a measure of the concentration of coliform

organism or E.coli in a water sample. It is defined as the reciprocal of
the smallest quantity of sample in ml which would give a positive
E.coli test. This index is now obsolete and now MPN – most probable
number is the one commonly used.

MPN – may be defined as that bacterial density which if it had been

actually present in the sample under examination, would more
frequently than any other have given the observed analytical result.

2. Physical Characteristics:

The physical characteristics of water would be examined atleast once a

week sample being drawn from representative points throughout should
not be so high as to offend the senses the sight, taste or smell of the
consumer.

MAV – Maximum Acceptable Values or

MAC – Maximum Acceptable Concentration
Turbidity Æ 5 units
Colours Æ 15 units
Odour No. Æ 3

3. Chemical Characteristics:
Drinking waster should not contain impurities in hazardous
concentrations be excessively corrosive or retain treatment substances
in excessive concentrations

91
DRINKING WATER STANDARDS:

Standards for water quality were originally a function of the public

health until 1970. With the passage of the Safe Drinking Water Act in
1974, the federal government through the Environmental Protection
Agency (EPA) was given the authority to set standards for drinking
water quality delivered by public water supplies.

92
 Waterborne diseases
Waterborne diseases are pathogenic microorganisms which are directly
transmitted when contaminated drinking water is consumed.
Contaminated drinking water used in the preparation of food can be the
source of foodborne disease through consumption of the same
microorganisms. According to the World Health Organization,
diarrheal disease accounts for an estimated 4.1% of the total DALY
global burden of disease and is responsible for the deaths of 1.8 million
people every year. It was estimated that 88% of that burden is
attributable to unsafe water supply, sanitation and hygiene and is
mostly concentrated on children in developing countries. Waterborne
disease can be caused by protozoa, viruses, bacteria, and intestinal
parasites

Protozoal infections

93
 Disease and Sources of Agent in Water General
Microbial Agent
Transmission Supply Symptoms

Abdominal
Protozoan discomfort, fatigue,
Amebiasis (hand- (Entamoeba Sewage, non-treated drinking weight loss,
to-mouth) histolytic) (Cyst-like water, flies in water supply diarrhea, gas pains
appearance) Fever, abdominal
pain, diarrhea

Flu-like symptoms,
watery diarrhea,
Collects on water filters and
Protozoan loss of appetite,
Cryptosporidiosis membranes that cannot be
(Cryptosporidium substantial loss of
(oral) disinfected, animal manure,
parvum) weight, bloating,
seasonal runoff of water.
increased gas,
stomach

cramps, nausea,
Protozoan parasite
Sewage, non-treated drinking vomiting, muscle
Cyclosporiasis (Cyclospora
water aches, low-grade
cayetanensis)
fever, and fatigue

Untreated water, poor

disinfection, pipe breaks, leaks, Diarrhea,
Protozoan (Giardia
Giardiasis (oral- groundwater contamination, abdominal
lamblia) Most
fecal) (hand-to- campgrounds where humans discomfort,
common intestinal
mouth) and wildlife use same source of bloating, gas and
parasite
water. Beavers and muskrats gas pains
act as a reservoir for Giardia.

94
Parasitic Infections

Disease and Microbial Sources of Agent in Water

General Symptoms
Transmission Agent Supply

Contaminated fresh water with

Schistosomiasis Rash or itchy skin. Fever, chills, cough,
Schistosoma certain types of snails that carry
(immersion) and muscle aches
schistosomes

dracanculus drinking water containing allergic reaction,urticarial rash, nausea,

dracunculiasis
medinensis infective cyclops vomiting, diarrhoea, asthmatic attack.

intestinal disturbances, neurologic

contaminate drinking water with
taeniasis solium Taenia solium manifestations, loss of weight,
eggs
cysticercosis

GIT disturbance, diarrhea, liver

contaminated drinking water with
fascioliasis fasciola enlargement, cholangitis, cholecystitis,
encysted metacercaria
obstructive jaundice.

hymenolepiasis hymenolepis contaminated drinking water with mild GIT symptoms, nervous
nana nana eggs manifestation

hyatid cyst press on bile ductand blood

echinococcus contaminated drinking water with
hyatidosis vessels, if it ruptured cause anaphylactic
granulosus eggs
shock.

multiceps contaminated drinking water with

coenurosis inreases intacranial tension
multiceps eggs

loefflers syndrome in lung, nausea,

ascaris contaminated drinking water with
ascraiasis vomiting, diarrhea, malnutrition,
lumbricoides eggs
underdevelopment,

entrobius contaminated drinking water with peri-anal itch, nervous irritability,

enterobiasis
vermicularis eggs hyperactivity and insomnia

95
 Morbility Mortality
Disease
(cases per year) (deaths per year)

1,500,000,000 100,000

Schistosomiasis 200,000,000 200,000

Bacterial infections

• Cholera - Vibrio cholerae bacteria - gastro-intestinal often

waterborne

• Botulism - Clostridium botulinum bacteria - gastro-intestinal

food/water borne; can grow in food

• Typhoid - Salmonella typhi bacteria - gastro-intestinal water/food

borne

• Dysentery - Shigella/Salmonella bacteria - gastro-intestinal

food/water

• Legionellosis

• Leptospirosis

Viral Infections

• Hepatitis A - Hepatitis A virus - gastro-intestinal water/food

borne

• Polio - polioviruses - gastro-intestinal exposure to untreated

• Caliciviruses, Astroviruses, Small Round Structured Virus

WHO's Guidelines for Drinking-water Quality, set up in Geneva, 1993, are

the international reference point for standard setting and drinking-water
safety.

96
nutrient/ Symbol/ Normally found in fresh Health based
substance formula water/surface guideline by the
water/ground water WHO

Aluminium Al 0,2 mg/l

Ammonia NH4 < 0,2 mg/l (up to 0,3 mg/l No guideline
in anaerobic waters)
Antimony Sb < 4 µg/l 0.005 mg/l
Arsenic As 0,01 mg/l
Asbestos No guideline
Barium Ba 0,3 mg/l
Berillium Be < 1 µg/l No guideline
Boron B < 1 mg/l 0,3 mg/l
Cadmium Cd < 1 µg/l 0,003 mg/l
Chloride Cl 250 mg/l
Chromium Cr+3, Cr+6 < 2 µg/l 0,05 mg/l
Colour Not mentioned
Copper Cu 2 mg/l
Cyanide CN- 0,07 mg/l
Dissolved oxygen O2 No guideline
Fluoride F < 1,5 mg/l (up to 10) 1,5 mg/l
Hardness mg/l No guideline
CaCO3
Hydrogen sulfide H2 S No guideline
Iron Fe 0,5 - 50 mg/l No guideline
Lead Pb 0,01 mg/l
Manganese Mn 0,5 mg/l
Mercury Hg < 0,5 µg/l 0,001 mg/l
Molybdenum Mb < 0,01 mg/l 0,07 mg/l
Nickel Ni < 0,02 mg/l 0,02 mg/l
Nitrate and nitrite NO3, NO2 50 mg/l total nitrogen
Turbidity Not mentioned
pH No guideline
Selenium Se < < 0,01 mg/l 0,01 mg/l
Silver Ag 5 – 50 µg/l No guideline
Sodium Na < 20 mg/l 200 mg/l
Sulfate SO4 500 mg/l
Inorganic tin Sn No guideline
TDS No guideline
Uranium U 1,4 mg/l
Zinc Zn 3 mg/l

97
 Organic compounds
Group Substance Formula Health based
guideline by the
WHO
Chlorinated alkanes Carbon tetrachloride C Cl4 2 µg/l
Dichloromethane C H2 Cl2 20 µg/l
1,1-Dichloroethane C2 H4 Cl2 No guideline
1,2-Dichloroethane Cl CH2 CH2 Cl 30 µg/l
1,1,1-Trichloroethane CH3 C Cl3 2000 µg/l
Chlorinated ethenes 1,1-Dichloroethene C2 H2 Cl2 30 µg/l
1,2-Dichloroethene C2 H2 Cl2 50 µg/l
Trichloroethene C2 H Cl3 70 µg/l
Tetrachloroethene C2 Cl4 40 µg/l
Aromatic Benzene C6 H6 10 µg/l
hydrocarbons Toluene C7 H8 700 µg/l
Xylenes C8 H10 500 µg/l
Ethylbenzene C8 H10 300 µg/l
Styrene C8 H8 20 µg/l
Polynuclear Aromatic Hydrocarbons (PAHs) C2 H3 N1 O5 P1 3 0.7 µg/l
Chlorinated Monochlorobenzene (MCB) C6 H5 Cl 300 µg/l
benzenes Dichlorobenzenes 1,2-Dichlorobenzene C6 H4 Cl2 1000 µg/l
(DCBs) (1,2-DCB)
1,3-Dichlorobenzene C6 H4 Cl2 No guideline
(1,3-DCB)
1,4-Dichlorobenzene C6 H4 Cl2 300 µg/l
(1,4-DCB)
Trichlorobenzenes (TCBs) C6 H3 Cl3 20 µg/l
Miscellaneous Di(2-ethylhexyl)adipate (DEHA) C22 H42 O4 80 µg/l
organic constituents Di(2-ethylhexyl)phthalate (DEHP) C24 H38 O4 8 µg/l
Acrylamide C3 H5 N O 0.5 µg/l
Epichlorohydrin (ECH) C3 H5 Cl O 0.4 µg/l
Hexachlorobutadiene (HCBD) C4 Cl6 0.6 µg/l
Ethylenediaminetetraacetic acid (EDTA) C10 H12 N2 O8 200 µg/l
Nitrilotriacetic acid (NTA) N(CH2COOH)3 200 µg/l
Organotins Dialkyltins R2 Sn X2 No guideline
Tributil oxide (TBTO) C24 H54 O Sn2 2 µg/l

98
 Pesticides
Substance Formula Health based guideline by
the WHO
Alachlor C14 H20 Cl N O2 20 µg/l
Aldicarb C7 H14 N2 O4 S 10 µg/l
Aldrin and dieldrin C12 H8 Cl6/ 0.03 µg/l

C12 H8 Cl6 O
Atrazine C8 H14 Cl N5 2 µg/l
Bentazone C10 H12 N2 O3 S 30 µg/l
Carbofuran C12 H15 N O3 5 µg/l
Chlordane C10 H6 Cl8 0.2 µg/l
Chlorotoluron C10 H13 Cl N2 O 30 µg/l
DDT C14 H9 Cl5 2 µg/l
1,2-Dibromo-3-chloropropane C3 H5 Br2 Cl 1 µg/l
2,4-Dichlorophenoxyacetic acid (2,4-D) C8 H6 Cl2 O3 30 µg/l
1,2-Dichloropropane C3 H6 Cl2 No guideline
1,3-Dichloropropane C3 H6 Cl2 20 µg/l
1,3-Dichloropropene CH3 CHClCH2 Cl No guideline
Ethylene dibromide (EDB) Br CH2 CH2 Br No guideline
Heptachlor and heptachlor epoxide C10 H5 Cl7 0.03 µg/l
Hexachlorobenzene (HCB) C10 H5 Cl7 O 1 µg/l
Isoproturon C12 H18 N2 O 9 µg/l
Lindane C6 H6 Cl6 2 µg/l
MCPA C9 H9 Cl O3 2 µg/l
Methoxychlor (C6H4OCH3)2CHCCl3 20 µg/l
Metolachlor C15 H22 Cl N O2 10 µg/l
Molinate C9 H17 N O S 6 µg/l
Pendimethalin C13 H19 O4 N3 20 µg/l
Pentachlorophenol (PCP) C6 H Cl5 O 9 µg/l
Permethrin C21 H20 Cl2 O3 20 µg/l
Propanil C9 H9 Cl2 N O 20 µg/l
Pyridate C19H23ClN2O2S 100 µg/l
Simazine C7 H12 Cl N5 2 µg/l
Trifluralin C13 H16 F3 N3 O4 20 µg/l
Chlorophenoxy 2,4-DB C10 H10 Cl2 O3 90 µg/l
herbicides (excluding Dichlorprop C9 H8 Cl2 03 100 µg/l
2,4-D and MCPA) Fenoprop C9H7Cl3O3 9 µg/l
MCPB C11 H13 Cl O3 No guideline
Mecoprop C10H11ClO3 10 µg/l
2,4,5-T C8 H5 Cl3 O3 9 µg/l

99
 Disinfectants and disinfectant by-products
Group Substance Formula Health based
guideline by the
WHO
Disinfectants Chloramines NHnCl(3-n), 3 mg/l
where
n = 0,
1 or 2
Chlorine Cl2 5 mg/l
Chlorine dioxide ClO2 No guideline
Iodine I2 No guideline
Disinfectant by- Bromate Br O3- 25 µg/l
products Chlorate Cl O3- No guideline
Chlorite Cl O2- 200 µg/l
Chlorophenols 2-Chlorophenol (2-CP) C6 H5 Cl O No guideline
2,4-Dichlorophenol (2,4-DCP) C6 H4 Cl2 O No guideline
2,4,6-Trichlorophenol (2,4,6-TCP) C6 H3 Cl3 O 200 µg/l
Formaldehyde HCHO 900 µg/l
MX (3-Chloro-4-dichloromethyl-5-hydroxy-2(5H)- C5 H3 Cl3 O3 No guideline
furanone)
Trihalomethanes Bromoform C H Br3 100 µg/l
Dibromochloromethane CH Br2 Cl 100 µg/l
Bromodichloromethane CH Br Cl2 60 µg/l
Chloroform CH Cl3 200 µg/l
Chlorinated acetic acids Monochloroacetic acid C2 H3 Cl O2 No guideline
Dichloroacetic acid C2 H2 Cl2 O2 50 µg/l
Trichloroacetic acid C2 H Cl3 O2 100 µg/l
Chloral hydrate (trichloroacetaldehyde) C Cl3 CH(OH)2 10 µg/l
Chloroacetones C3 H5 O Cl No guideline
Halogenated acetonitriles Dichloroacetonitrile C2 H Cl2 N 90 µg/l
Dibromoacetonitrile C2 H Br2 N 100 µg/l
Bromochloroacetonitrile CH Cl2 CN No guideline
Trichloroacetonitrile C2 Cl3 N 1 µg/l
Cyanogen chloride Cl CN 70 µg/l
Chloropicrin C Cl3 NO2 No guideline

100
 WATER-BORNE DISEASES
Waterborne diseases are pathogenic microorganisms which are directly
transmitted when contaminated drinking water is consumed.
Contaminated drinking water used in the preparation of food can be the
source of foodborne disease through consumption of the same
microorganisms. According to the World Health Organization,
diarrheal disease accounts for an estimated 4.1% of the total DALY
global burden of disease and is responsible for the deaths of 1.8 million
people every year. It was estimated that 88% of that burden is
attributable to unsafe water supply, sanitation and hygiene and is
mostly concentrated on children in developing countries.

Waterborne disease can be caused by protozoa, viruses, bacteria, and

intestinal parasites

Water is one of the chief vehicles of gastrointestinal diseases and the

provision of a safe water supply is one of the first tasks to be
undertaken in the introduction of environmental sanitation. The task is
not an easy one. The bacterial and viral diseases carried by water
include

Enteric fever: Typhoid fever, also known as enteric fever, is an

illness caused by the bacterium Salmonella typhi. Common worldwide,
it is transmitted by ingestion of food or water contaminated with feces
from an infected person. The bacteria then multiply in the blood stream
of the infected person and are absorbed into the digestive tract and

eliminated with the waste

Fig. Salmonella typhi

Dysentery: Dysentery (formerly known as flux or the bloody flux) is

101
the term for tenesmus (painful straining to pass stool), cramping, and
frequent, small-volume severe diarrhea associated with blood in the
feces. Symptoms frequently associated with dysentery include fever
and malaise.

Dysentery has many causes, including cancer, but is typically

associated with infection caused by the ingestion of food or water
containing micro-organisms which cause significant inflammation of
the intestinal lining. There are two major types: shigellosis, which is
caused by one of several types of Shigella bacteria; and amoebic
dysentery, caused by the amoeba Entamoeba histolytica. Kiyoshi Shiga
discovered the dysentery bacteria in 1898. Amoebic dysentery

Amoebic dysentery is transmitted through contamination of drinking

water, and is well known as a "traveler's dysentery" because of its
prevalence in developing nations, or "Montezuma's Revenge" although
it is occasionally seen in industrialized countries. Liver infection and
subsequent amoebic abscesses can occur. It is caused mainly by the
protozoan Entamoeba histolytica. Amoebic dysentery can be treated
with metronidazole

Cholera (or Asiatic cholera or epidemic cholera) is a severe diarrheal

disease caused by the bacterium Vibrio cholerae. Transmission to
humans is by ingesting contaminated water or food. The major
reservoir for cholera was long assumed to be humans, but some
evidence suggests that it is the aquatic environment. It is extremely
deadly.

V. cholerae is a Gram negative bacterium which produces cholera

toxin, an enterotoxin, whose action on the mucosal epithelium lining of
the small intestine is responsible for the characteristic massive diarrhea
of the disease. In its most severe forms, cholera is one of the most
rapidly fatal illnesses known: A healthy person may become
hypotensive within an hour of the onset of symptoms and may die
within 2-3 hours if no treatment is provided. More commonly, the
disease progresses from the first liquid stool to shock in 4-12 hours,
with death following in 18 hours to several days without rehydration
treatment.

102
Legionnaires’ diseases:

Infectious hepatitis:

Gastroenteritis

Conjunctivitis due to Chlamydia trachomalis has been transmitted in

inadequately chlorinated swimming pools.

Occasional outbreaks of Weil’s disease

Tularemia

John Snow (1855) in his study of cholera was first to bring conclusive
evidence was the first to bring conclusive evidence of the waterborne
carriage of the disease.

103
 PURIFICATION OF WATER

Water purification is the process of removing contaminants from a

raw water source. The goal is to produce water for a specific purpose
with a treatment profile designed to limit the inclusion of specific
materials; most water is purified for human consumption (drinking
water). Water purification may also be designed for a variety of other
purposes and water purified to meet the requirements of medical,
pharmacology, chemical and industrial applications. Methods include,
but are not limited to: ultra violet light, filtration, water softening,
reverse osmosis, ultrafiltration, molecular stripping, deionization, and
carbon treatment.

Water purification may remove particulate sand; suspended particles of

organic material; Parasites, Giardia; Cryptosporidium; bacteria; algae;
virus; fungi; etc. Minerals calcium, silica, magnesium, etc., and Toxic
metals lead; copper; chromium; etc. Some purification may be elective
in its inclusion in the purification process; examples, smell (hydrogen
sulfide remediation), taste (mineral extraction), and appearance (iron
incapsulation).

Governments usually dictate the quality standards for drinking water

quality these standards will require minimum / maximum setpoints for
the extraction of contaminants and the inclusion of control elements
that produce potable drinking water. Quality standards in the United
States require specific amounts of disinfectant (example, residual
chlorine content) in the water after it leaves the WTP (Water Treatment
Plant), at the end of the treatment process to reduce the risk of re-
contamination while the water is in the distribution system.

Ground water (usually supplied as well water) is typically a more

economical choice than surface water as a source for drinking water, as
it is inherently pre-filtered, by the aquifer from which it is extracted.
Over large areas of the world, aquifers are recharged as part of the
hydrologic cycle, and their water is a renewable resource. In more arid

104
regions, water from an aquifer will have a limited output and can take
thousands of years to recharge. Surface water; (rivers, lakes, streams) is
locally more abundant where subsurface formations do not function as
aquifers; however, ground water is far more abundant than the more-
visible surface water. Surface water is a typical raw water source used
to make drinking water where it is abundant, ground water is
unavailable or poor quality, and however, it is much more exposed to
human activity and its byproducts. As a water source it is carefully
monitored for the presence of a variety of contaminants by the WTP
operators.

It is not possible to tell whether water is safe to drink just by looking at

it. Simple procedures such as boiling or the use of a household charcoal
filter are not sufficient for treating all the possible contaminants that
may be in water from an unknown source. Even natural spring water;
considered safe for all practical purposes in the 1800s; and must now
be tested before determining what kind of treatment is needed.
Laboratory analysis will define the contaminants in the water sample,
with both qualitative and quantitative measurements. Lab analysis,
while expensive, it is the only way you will be able to obtain the bench
mark information necessary for establishment of a purification process,
methodology for purification.

1. Deep ground water: The water emerging from some deep

groundwaters may have fallen as rain many decades, hundreds,
thousands or in some cases millions of years ago. Soil and rock
layers naturally filter the ground water to a high degree of clarity
before it is pumped to the treatment plant. 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 such as
Campylobacter or the pathogenic protozoa Cryptosporidium and
Giardia) are typically absent, but the water typically is rich in
dissolved solids, especially carbonates and sulphates 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

105
 or manganese content of this water to make it pleasant for
drinking, cooking, and laundry use. Disinfection is also required.
Where groundwater recharge is practised, it is equivalent to
lowland surface waters for treatment purposes.
2. Shallow groundwaters: Water emerging from shallow
groundwaters is usually abstracted from wells or boreholes. The
bacteriological quality can be variable depending on the nature of
the catchment. A variety of soluble materials may be present
including (rarely) potentially toxic metals such as zinc and
copper. Arsenic contamination of groundwater is a serious
problem in some areas, notably from shallow wells in Bangladesh
and West Bengal in the Ganges Delta.
3. Upland lakes and reservoirs: Typically located in the
headwaters of river systems, upland reservoirs are usually sited
above any human habitation and may be surrounded by a
protective zone to restrict the opportunities for contamination.
Bacteria and pathogen levels are usually low, but some bacteria,
protozoa or algae will be present. Where uplands are forested or
peaty, humic acids can colour the water. Many upland sources
have low pH which requires adjustment.
4. 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.
5. 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 vapour.
6. 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.

Pre-treatment

1. Pumping and containment - The majority of water must be

pumped from its source or directed into pipes or holding tanks.
To avoid adding contaminants to the water, this physical
infrastructure must be made from appropriate materials and

106
 constructed so that accidental contamination does not occur.
2. Screening - The first step in purifying surface water is to remove
large debris such as sticks, leaves, trash and other large particles
which may interfere with subsequent purification steps. Most
deep Groundwater does not need screening before other
purification steps.
3. Storage - Water from rivers may also be stored in bankside
reservoirs for periods between a few days and many months to
allow natural biological purification to take place. This is
especially important if treatment is by slow sand filters. Storage
reservoirs also provide a buffer against short periods of drought
or to allow water supply to be maintained during transitory
pollution incidents in the source river.
4. Pre-conditioning - Many waters rich in hardness salts are treated
with soda-ash (Sodium carbonate) to precipitate calcium
carbonate out utilising the common ion effect.
5. Pre-chlorination - In many plants the incoming water was
chlorinated to minimise the growth of fouling organisms on the
pipe-work and tanks. Because of the potential adverse quality
effects (see Chlorine below), this has largely been discontinued.

Widely varied techniques are available to remove the fine solids,

micro-organisms and some dissolved inorganic and organic materials.
The choice of method will depend on the quality of the water being
treated, the cost of the treatment process and the quality standards
expected of the processed water

pH adjustment

Distilled water has an average pH of 7 (neither alkaline or acidic) and

sea water has an average pH of 8.3 (slightly alkaline). If the water is
acidic (lower than 7), lime or soda ash is added to raise the pH. Lime is
the more common of the two additives because it is cheaper, but it also
adds to the resulting water hardness. Making the water slightly alkaline
ensures that coagulation and flocculation processes work effectively
and also helps to minimise the risk of lead being dissolved from lead
pipes and lead solder in pipe fittings.

107
FLOCUATION: is a process in which we first clarify the water.
Clarifying means removing any turbidity or colour so that the water is
sparklingly clear and colourless. Clarification is done by causing a
precipitate to form in the water. Initially the precipitate forms as very
small particles but as the water is gently stirred, these particles stick
together to form bigger particles. We can say that the small particles
coagulate; this process is sometimes called flocculation. Many of the
small particles that were originally present in the raw water absorb onto
the surface of these small precipitate particles and so get incorporated
into the larger particles that coagulation produces. In this way the
coagulated precipitate takes most of the suspended matter out of the
water and is then filtered of, generally by passing the mixture through a
coarse sand filter or sometimes through a mixture of sand and
granulated anthracite (high quality coal). Anthracite with its high
carbon content is able to absorb much of the organic matter present in
solution and this can remove odour and taste from the water. A
precipitate that is widely used to clarify water is iron (III) hydroxide.
This is formed first by adjusting (if necessary) the pH of the incoming
water to above 7 (by adding lime or sodium hydroxide), then by adding
a solution of an iron (III) compound such as iron (III) chloride. Iron
(III) hydroxide is extremely insoluble and forms even at a pH as low as
7. Aluminium hydroxide is also widely used as the flocculating
precipitate.

Sedimentation

Water exiting the flocculation basin may enter the sedimentation basin,
also called a clarifier or settling basin. It is a large tank with slow flow,
allowing floc to settle to the bottom. The sedimentation basin is best
located close to the flocculation basin so the transit between does not
permit settlement or floc break up. Sedimentation basins can be in the
shape of a rectangle, where water flows from end to end, or circular
where flow is from the center outward. Sedimentation basin outflow is
typically over a weir so only a thin top layer-furthest from the
sediment-exits. The amount of floc that settles out of the water is
dependent on the time the water spends in the basin and the depth of
the basin. The retention time of the water must therefore be balanced

108
against the cost of a larger basin. The minimum clarifier retention time
is normally 4 hours. A deep basin will allow more floc to settle out than
a shallow basin. This is because large particles settle faster than smaller
ones, so large particles bump into and integrate smaller particles as they
settle. In effect, large particles sweep vertically though the basin and
clean out smaller particles on their way to the bottom.
As particles settle to the bottom of the basin a layer of sludge is formed
on the floor of the tank. This layer of sludge must be removed and
treated. The amount of sludge that is generated is significant, often 3%-
5% of the total volume of water that is treated. The cost of treating and
disposing of the sludge can be a significant part of the operating cost of
a water treatment plant. The tank may be equipped with mechanical
cleaning devices that continually clean the bottom of the tank or the
tank can be taken out of service when the bottom needs to be cleaned.

Filtration

After separating most floc, the water is filtered as the final step to
remove remaining suspended particles and unsettled floc. The most
common type of filter is a rapid sand filter. Water moves vertically
through sand which often has a layer of activated carbon or anthracite
coal above the sand. The top layer removes organic compounds, which
contribute to taste and odour. The space between sand particles is larger
than the smallest suspended particles, so simple filtration is not enough.
Most particles pass through surface layers but are trapped in pore
spaces or adhere to sand particles. Effective filtration extends into the
depth of the filter. This property of the filter is key to its operation: if
the top layer of sand were to block all the particles, the filter would
quickly clog.
To clean the filter, water is passed quickly upward through the filter,
opposite the normal direction (called backflushing or backwashing) to
remove embedded particles. Prior to this, compressed air may be blown
up through the bottom of the filter to break up the compacted filter
media to aid the backwashing process; this is known as air scouring.
This contaminated water can be disposed of, along with the sludge
from the sedimentation basin, or it can be recycled by mixing with the
raw water being entering the plant. Some water treatment plants

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employ pressure filters. These work on the same principle as rapid
gravity filters differing in that the filter medium is enclosed in a steel
vessel and the water is forced through it under pressure.

Membrane filtration: is essentially a thin film of synthetic polymer

through which there are pores of fairly uniform size. This filters water
as it flows through.

ADVANTAGES: Filter out much smaller particles than paper and sand
filters can Filter out virtually all particles larger than their specified
pore sizes They are quite thin and so liquids flow through them fairly
rapidly. They are reasonably strong and so can withstand pressure
differences across them of typically 2-5 atmospheres. They can be
cleaned (back flushed) and reused.

Membrane filters are widely used for filtering both drinking water and
sewage (for reuse). For drinking water membrane filters can remove
virtually all particles larger than 0.2 um including Giardia and
cryptosporidium. Membrane filters is an effective form of tertiary
treatment when it is desired to reuse the water for industry or for
limited domestic purposes or before discharging the water into a river
that is used by towns further downstream. Is widely used in industry,
particularly for beverage preparation (including bottled water).
However no filtration can remove substances that are actually dissolved
in the water such as phosphorus and nitrates and heavy metal ions.

Slow sand filters

Slow sand filters may be used where there is sufficient land and space.
These rely on biological treatment processes for their action rather than
physical filtration. Slow sand filters are carefully constructed using
graded layers of sand with the coarsest at the top and finest at the base.
Drains at the base convey treated water away for disinfection. Filtration
depends on the development of a thin biological layer on the surface of
the filter. An effective slow sand filter may remain in service for many
weeks or even months if the pre-treatment is well designed and
produces an excellent quality of water which physical methods of
treatment rarely achieve.

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Ultrafiltration

Ultrafiltration membranes are a relatively new development; they use

polymer film with chemically formed microscopic pores that can be
used in place of granular media to filter water effectively without
coagulants. The type of membrane media determines how much
pressure is needed to drive the water through and what sizes of micro-
organisms can be filtered out.

Active coal can fulfill this role.

DISINFECTION:

1. Chlorination- The most common disinfection method is some

form of chlorine or its compounds such as Chloramine or
chlorine dioxide. Chlorine is a strong oxidant that kills many
micro-organisms.
Because chlorine is a toxic gas, there is a danger of a release
associated with its use. This problem is avoided by the use of
sodium hypochlorite, which is either a relatively inexpensive
solid that releases free chlorine when dissolved in water or a
liquid (bleach) that is typically generated on site using common
salt and high voltage DC. Handling the solid, however, requires
greater routine human contact through opening bags and pouring
than the use of gas cylinders which are more easily automated.
The generation of liquid sodium hypochlorite is both inexpensive
and safer than the use of gas or solid chlorine. Both disinfectants
are widely used despite their respective drawbacks. One
drawback to using chlorine gas or sodium hypochlorite is that
they react with organic compounds in the water to form
potentially harmful chemical by-products Trihalomethanes
(THMs) and haloacetic acids (HAAs), both of which are
carcinogenic in large quantities and regulated by the U.S.
Environmental Protection Agency (EPA). The formation of
THMs and haloacetic acids is minimized by effective removal of
as many organics from the water as possible prior to chlorine
addition. Although chlorine is effective in killing bacteria, it has
limited effectiveness against protozoans that form cysts in water.

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 (Giardia lamblia and Cryptosporidium, both of which are
pathogenic).
2. Chlorine dioxide is another fast-acting disinfectant. It is,
however, rarely used, because it may create excessive amounts of
chlorate and chlorite, both of which are regulated to low
allowable levels. Chlorine dioxide also poses extreme risks in
handling: not only is the gas toxic, but it may spontaneously
detonate upon release to the atmosphere in an accident.
3. Chloramines are another chlorine-based disinfectant. Although
chloramines are not as strong of an oxidant or provide a reliable
residual, as compared to chlorine gas or sodium hypochlorite,
they are less prone to form THMs or haloacetic acids. It is
possible to convert chlorine to Chloramine by adding ammonia to
the water along with the chlorine: The chlorine and ammonia
react to form Chloramine. Water distribution systems disinfected
with chloramines may experience nitrification, wherein ammonia
is used a nitrogen source for bacterial growth, with nitrates being
generated as a byproduct.
4. Ozone (O3) is a relatively unstable molecule "free radical" of
oxygen which readily gives up one atom of oxygen providing a
powerful oxidising agent which is toxic to most water borne
organisms. It is a very strong, broad spectrum disinfectant that is
widely used in Europe. It is an effective method to inactivate
harmful protozoans that form cysts. It also works well against
almost all other pathogens. Ozone is made by passing oxygen
through ultraviolet light or a "cold" electrical discharge. To use
ozone as a disinfectant, it must be created on site and added to the
water by bubble contact. Some of the advantages of ozone
include the production of relatively fewer dangerous by-products
(in comparison to chlorination) and the lack of taste and odor
produced by Ozonation. Although fewer by-products are formed
by Ozonation, it has been discovered that the use of ozone
produces a small amount of the suspected carcinogen Bromate,
although little Bromine should be present in treated water.
Another one of the main disadvantages of ozone is that it leaves
no disinfectant residual in the water. Ozone has been used in
drinking water plants since 1906 where the first industrial

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 Ozonation plant was built in Nice, France. The U.S. Food and
Drug Administration has accepted ozone as being safe; and it is
applied as an anti-microbiological agent for the treatment,
storage, and processing of foods.
5. UV radiation (light) is very effective at inactivating cysts, as long
as the water has a low level of colour so the UV can pass through
without being absorbed. The main disadvantage to the use of UV
radiation is that, like ozone treatment, it leaves no residual
disinfectant in the water.
Because neither ozone nor UV radiation leaves a residual
disinfectant in the water, it is sometimes necessary to add a
residual disinfectant after they are used. This is often done
through the addition of chloramines, discussed above as a
primary disinfectant. When used in this manner, chloramines
provide an effective residual disinfectant with very little of the
negative aspects of chlorination.

Other water purification techniques

Other popular methods for purifying water, especially for local private
supplies are listed below. In some countries some of these methods are
also used for large scale municipal supplies. Particularly important are
distillation (de-salination of seawater) and reverse osmosis.

1. Boiling: Water is heated hot enough and long enough to

inactivate or kill micro-organisms that normally live in water at
room temperature. Near sea level, a vigorous rolling boil for at
least one minute is sufficient. At high altitudes (greater than two
kilometers or 5000 feet) three minutes is recommended. In areas
where the water is "hard" (that is, containing significant dissolved
calcium salts), boiling decomposes the bicarbonate ions, resulting
in partial precipitation as calcium carbonate. This is the "fur" that
builds up on kettle elements, etc., in hard water areas. With the
exception of calcium, boiling does not remove solutes of higher
boiling point than water and in fact increases their concentration
(due to some water being lost as vapour). Boiling does not leave
a residual disinfectant in the water. Therefore, water that has been
boiled and then stored for any length of time may have acquired

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 new pathogens.
2. Carbon filtering: Charcoal, a form of carbon with a high surface
area, absorbs many compounds including some toxic compounds.
Water passing through activated charcoal is common in
household water filters and fish tanks. Household filters for
drinking water sometimes contain silver to release silver ions
which have an anti-bacterial effect.
3. Distillation involves boiling the water to produce water vapour.
The vapour contacts a cool surface where it condenses as a liquid.
Because the solutes are not normally vaporised, they remain in
the boiling solution. Even distillation does not completely purify
water, because of contaminants with similar boiling points and
droplets of unvaporised liquid carried with the steam. However,
99.9% pure water can be obtained by distillation. Distillation
does not confer any residual disinfectant and the distillation
apparatus may be the ideal place to harbour Legionnaires'
disease.
4. Reverse osmosis: Mechanical pressure is applied to an impure
solution to force pure water through a semi-permeable
membrane. Reverse osmosis is theoretically the most thorough
method of large scale water purification available, although
perfect semi-permeable membranes are difficult to create. Unless
membranes are well-maintained, algae and other life forms can
colonise the membranes.
5. Ion exchange: Most common ion exchange systems use a zeolite
resin bed to replace unwanted Ca2+ and Mg2+ ions with benign
(soap friendly) Na+ or K+ ions. This is the common water
softener.
6. Electrodeionization: Water is passed between a positive
electrode and a negative electrode. Ion selective membranes
allow the positive ions to separate from the water toward the
negative electrode and the negative ions toward the positive
electrode. High purity deionized water results. The water is
usually passed through a reverse osmosis unit first to remove
non-ionic organic contaminants.
7. The use of iron in removing arsenic from water. See Arsenic
contamination of groundwater Water purification solutions

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Pharmaceutical Microbiology
Pharmaceutical microbiology is the part of industrial microbiology
that is responsible for creating medications. Pharmaceutical
Microbiology is an applied science discipline very relevant to infection
and contamination control. It draws upon understandings of
mechanisms of antimicrobial action and their relationship to resistance,
hygiene in the healthcare environment, and microbial pathogenicity and
behaviour.

Good Manufacturing Practice or GMP (also referred to as 'cGMP' or

'current Good Manufacturing Practice') is a term that is recognized
worldwide for the control and management of manufacturing and
quality control testing of foods and pharmaceutical products.

Good manufacturing practice comprises that part of quality assurance

aimed at ensuring that a product is consistently manufactured to a
quality appropriate for its intended to a quality appropriate for its
intended use.

GMP requires that a manufacturing process is fully defined before it is

initiated and that all necessary facilities are provided. In practice, this
means that personnel must be adequately trained.

Æ Suitable premises and equipments used

Æ Correct materials used

Æ Approved procedures adopted

Æ Suitable storage & transport facilities and

Æ Appropriate records

The quality of a pharmaceutical product depends on the degree of care

taken in its preparation. Final checks carried out on the finished

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product are useful in confirming that the correct ingredients have been
used and that the materials have been correctly processed.

It is however essential that proper in-process control is exercised and

that it is adequately documented to provide reliable evidence that the
control procedures have been followed.

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 1. Equipments of good 2. Correct Choice of
design and properly Cleaning Equipment
maintained Regularly monitored

4. Manufacturing
Premises of Good Design
3. Quality control of raw & Regularly Monitored
material

5. Quality control of
finished product

6. Written procedures & 7. Stained staff wearing

other documentation protective clothing

8.Quality control of
packaging

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GMP HISTORY:

The term Good Manufacturing Practice was introduced to regulate

manufacturing and packaging operations in the pharmaceutical
industry. Until the mid 1960s, operating procedures for the
manufacture of pharmaceuticals consisted of the formulae and the basic
methods of making products. The written procedures were often
concise and often relied on the individual operators’ skill and
experience in actually producing the product. As batches of
pharmaceutical products increased in number and size, it became
apparent that the operating procedures were inadequate to produce
consistent and reliable products. Much attention had focused on the
purity of the drug, particularly as many had been derived from natural
products. Active ingredients with greater potencies were also being
used in formulating new products.

Pharmacopoeias and codices specified formulae for mixtures and other

preparations but gave little detailed information on the methods of
preparation. The factors affecting processing and packaging
procedures were becoming more apparent and the need for appropriate
guidelines was evident. Guide to the GMP was prepared and compiled
by the Medicines Inspectorate of the Department of Health & Social
Security in consultation with other interested bodies.

The first edition was published in the year 1971. It’s purpose was to
recommend steps that should be taken as necessary and appropriate by
manufacturers of medicinal products with the object of ensuring that
their products were of the nature and quality intended. The second
guide was published in 1977 and the third in 1983.

In the USA, GMP regulations were developed by the Food and Drug
Administration and issued in the US Code of Federal Regulations.
GMP regulations present the minimum requirements to be met by
industry for the manufacture, processing, packaging and storage of
human and veterinary drugs. Under the Federal Food, Drug &
Cosmetic Act, a drug is considered to be adulterated unless the methods
used in its manufacture, processing, and packaging and storage as well
as the facilities and controls used, confirm to current GMP.

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The drug should meet the safety requirements of the Act and should
have the identity and strength to meet the quality and purity
characteristics that it is represented to have. Manufacturing
authorizations are required by all pharmaceutical manufacturers.

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REQUIREMENTS OF GMP & QUALITY MANAGEMENT

The holder of a manufacturing authorization must produce medicinal

products in a manner that ensures preparations are fit for their intended
use, comply with the requirements of the marketing authorization and
do not place patients at risk due to inadequate safety, quality of
efficacy. To achieve the quality objective reliably there must be a
comprehensively designed and correctly implemented system of quality
assurance incorporating GMP and thus quality control. The basic
concepts of quality assurance, GMP and quality control are inter-
related.

Quality Assurance:

It is a wide ranging concept that includes all matters that individually or

jointly influence the quality of a product. It is the sum total of the
organized arrangements made with the object of ensuring that
medicinal products are of the quality required for their intended use.
Therefore quality assurance incorporates Good Manufacturing Practice
(GMP) in addition to other factors.

Quality Control:

Quality control is that part of Good Manufacturing Practice (GMP) that

is concerned with sampling, specifications and testing and with the
organization, documentation and release procedures that ensure that the
necessary and relevant tests are actually carried out and that materials
are not released for use or products released for sale or supply until
their quality has been judged to be satisfactory.

Validation:

Validation is the action of proving in accordance with the principles of

GMP that any procedure, process, equipment, material, activity or
system leads to the expected results. In addition to being an essential
component of quality assurance, GMP is concerned with both
production and quality control.

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 PRINCIPAL REQUIRMENTS OF GOOD MANUFACTURING
PRACTICES GMP
1. Manufacturing processes are defined and capable of
producing products of suitable quality and specifications
2. Critical steps of manufacturing processes and significant
changes to the process are validated
3. Necessary facilities are provided including qualified and
trained personnel, adequate premises and space, correct
materials, containers and labels, approved procedures and
instructions, suitable storage areas and transport.
4. Instructions and procedures are clearly written
5. Operators are appropriately trained
6. Records are made demonstrating that all procedures and
instructions were followed and that the quality and quantity
of the product was as expected
7. Records of manufacture enabling the history of a batch to
be traced should be retained
8. Distribution minimizes any risk to product quality
9. A system is available to recall any batch of product
10. Complaints about marketed products are examined
and measures taken to prevent recurrences, if appropriate
11. A clear of the product to be manufactured
12. Raw materials to be used
13. Factors affecting formulations
14. Standardization of the procedure
15. Quality of the end product
16. Efficacy of the end product
17. Lab-animal studies, Good record of the production
and marketing
18. After-marketing surveys and preventive measures
19. Studies on long-term effects

GMP – It involves the following

• Initiating GMP
• Installing Systems

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 • Training Staff
• Maintaining Building and Equipments.

These are very costly but it should be considered a long-term

investment. The cost may be negligible compared to anything other
than strict adherence to GMP Regulatory Authorities would not allow
the product to be marketed; a defective product could have serious
consequence for the patient receiving it and the company may face
serious repercussions. Therefore GMP in a pharmaceutical industry is
an intrinsic part of technical strategy and business strategy.

Personnel: The establishment and maintenance of a competent system

of quality assurance and the correct manufacture of medicinal products
relies upon personnel. There must be sufficient qualified personnel to
carry out all the tasks that are the responsibility of the manufacturer.
Individual responsibilities should be clearly understood by the
individuals and recorded. All personnel should be aware of the
principles of GMP that affect them and received initial and continuing
training, including hygiene instructions relevant to their needs.
Training records should be kept.

Three Key Personnel are specified:

1. The head of production

2. The head of quality control &
3. Qualified staff

The quality controller and the production manager should be different

persons acting independently but jointly responsible to the quality of
the products. The qualified staff is responsible for other duties which
are not performed either by the head of production or the head of
quality control. These duties are to ensure that batches including these
imported from outside have been produced, tested and checked in
accordance with directives and marketing authorization. The qualified
persons must also certify that each production batch satisfy the
provisions of GMP.

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 1. All personnel should have a medical examination on
recruitment. Persons with potentially infectious diseases or
open lesions on exposed surfaces should not be involved in
the manufacture of medicinal products.
2. Appropriate protective garments should be worn in
manufacturing areas. Care should be taken to avoid direct
contact between the operator’s hand and the exposed
product as well as any part of the equipment that comes
into contact with the product.
3. Eating, drinking, chewing or smoking, the storage of food,
drink or smoking materials or personal medications should
be prohibited in the manufacturing area

PREMISES & EQUIPMENT:

Premises and Equipment must be located, designed, constructed,

adapted and maintained to suit the operations to be carried out. The
layout and design of premises and equipment should minimise the risks
of errors and permit effective cleaning and maintenance in order to
avoid cross-contamination, build up dust or dirt, in general any adverse
effect on the quality of products. Premises should be designed, built
and maintained to suit the operations being undertaken. GMP makes
recommendations on the design of the production areas, storage areas,
quality control areas and ancillary areas for rest and refreshments.

Equipments should be designed and installed to suit the process being

carried out. It must be cleaned, maintained and repaired without
causing any contamination to materials or products.

DOCUMENTATION:

1. A good documentation is an essential requirement of the

quality assurance system.
2. Clearly written documentation avoids errors from spoken
communication and allows tracing of batch history.
3. Specifications, manufacturing formulae and instructions,
procedures and records must be free from errors and
available in writing.

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 4. The legibility of documents is important
5. Documentation should be available to give details of
a) Specification for starting materials, intermediate
and bulk materials, finished products and
packaging materials
b) Manufacturing formula, processing and packaging
instructions
c) Procedures
d) Records
6. All documentations should be unambiguous, regularly
reviewed and updated where necessary.
7. Records must be completed at the time of action and kept
until one year after the expiry of the final product
8. When entries have be altered, the alterations should be
signed and dated and a reason given. The alteration should
be made in such a way that the original entry is still legible
9. When electronic data processing systems are used, access
should be limited by a password or other means; only
authorized persons should be able to enter or modify data
10. Electronically stored documentation must be readily
available at all times and be protected by a system of back
up transfers
11. Records must be kept of changes to or deletion from,
electronically stored data

PRODUCTION:

Production operations must follow clearly defined procedures.

1. Operations must comply with the principles of GMP in order to

produce products of the stipulated quality and be in accordance
with their manufacturing and marketing authorizations.
2. The measures taken to prevent cross-contamination of starting
materials and products should be regularly assessed.
3. Validation studies should reinforce GMP and be conducted in
accordance with defined procedures
4. Guidelines are given for the purchase, delivery, labeling and
storage of starting materials

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5. The purchase, handling and control of primary and printed
packaging materials should be accorded attention similar to that
given to starting materials.
6. Processing and packaging operations should be conducted in
accordance with the defined procedures.
7. Finished products should be held in quarantine until their final
release under conditions established by the manufacturer.
8. Rejected materials and products should be clearly marked as such
and stored separately in restricted areas. They should either be
returned to the suppliers or where appropriate, reprocesses or
destroyed.

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 Production of antibiotics
Industrial production techniques

Antibiotics are produced industrially by a process of fermentation,

where the source microorganism is grown in large containers (100,000–
150,000 liters or more) containing a liquid growth medium. Oxygen
concentration, temperature, pH and nutrient levels must be optimal, and
are closely monitored and adjusted if necessary. As antibiotics are
secondary metabolites, the population size must be controlled very
carefully to ensure that maximum yield is obtained before the cells die.
Once the process is complete, the antibiotic must be extracted and
purified to a crystalline product. This is simpler to achieve if the
antibiotic is soluble in organic solvent. Otherwise it must first be
removed by ion exchange, adsorption or chemical precipitation.

Strains used for production

Microorganisms used in fermentation are rarely identical to the wild

type. This is because species are often genetically modified to yield the
maximum amounts of antibiotics. Mutation is often used, and is
encouraged by introducing mutagens such as ultraviolet radiation, x-
rays or certain chemicals. Selection and further reproduction of the
higher yielding strains over many generations can raise yields by 20-
fold or more. Another technique used to increase yields is gene
amplification, where copies of genes coding for proteins involved in the
antibiotic production can be inserted back into a cell, via vectors such
as plasmids. This process must be closely linked with retesting of
antibiotic production and effectiveness.

The use of fermentation is an important process in the industry. Though

fermentation can have stricter definitions, when speaking of it in
Industrial fermentation, it more loosely refers to the breakdown of
organic substances and re-assembly into other substances. Somewhat
paradoxically, fermenter culture in industrial capacity often refers to
highly oxygenated and aerobic growth conditions, whereas
fermentation in the biochemical context is a strictly anaerobic process.

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Pharmaceuticals and Biotech Industry

There are 5 major groups of commercially important Fermentation

1. Microbial cells or Biomass as the product: Eg. Bakers Yeast,

Lactic acid bacillus, Bacillus sp.
2. Microbial Enzymes: Catalase, Amylase, Protease, Pectinase,
Glucose isomerase, Cellulase, Hemicellulase, Lipase, Lactase,
Streptokinase etc.
3. Microbial metabolites :
1. Primary metabolites – Ethanol, Citric acid, Glutamic acid,
Lysine, Vitamins, Polysaccharides etc.
2. Secondary metabolites: All antibiotic fermentation
4. Recombinant products : Insulin, HBV, Interferon, GCSF,
Streptokinase
5. Biotransformation: Eg. Phenyl acetyl carbinol, Steroid
Biotransformation

Nutrient sources for industrial fermentation

Medium for Industrial Fermentations

Any Microbe requires Water, Oxygen, Energy source, Carbon source,

Nitrogen source and Micronutrients for the growth.

Carbon & Energy source + Nitrogen source + O2 + other requirements

→ Biomass + Product + byproducts + CO2 + H2O + heat

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Nutrient Raw material

Carbon

Glucose Corn sugar, Starch, Cellulose

Sucrose Sugarcane, Sugar beet molasses

Lactose Milk whey

Fats Vegetable oils

Hydrocarbons Petroleum fractions

Nitrogen

Protein Soybean meal, Corn steep liquor, Distillers' solubles

Pure ammonia or ammonium salts

Ammonia
Urea

Nitrate Nitrate salts

Phosphorus
Phosphate salts
source

Trace elements: Fe, Zn, Cu, Mn, Mo, Co

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Antifoaming agents: Esters, Fatty acids, Silicones, Sulphonates,
Polypropylene

Buffers: Calcium carbonate, Phosphates

Growth factors: Some microorganisms cannot synthesize the required

cell components themselves and need to be supplemented: E.g.
Thiamine, Biotin, Calcium pentothenate

Precursors: Directly incorporated into the desired product: Phenyl

ethylamine into Benzyl penicillin, Phenyl acetic acid into Penicillin G

Inhibitors: To get the specific products: e.g. Sodium barbital for

Rifamycin

Inducers: Majority of the enzymes are inducible and are synthesized in

response of inducers: e.g. Starch for Amylases, Maltose for
Pollulanase, Pectin for Pectinase

Chelators: Chelators are the chemicals used to avoid the precipitation

of metal ions. Chelators like EDTA, Citric acid, Polyphosphates are
used in low concentrations.

A vaccine is an antigenic preparation used to establish immunity to a

disease. The term derives from Edward Jenner's use of cowpox
("vacca" means cow in Latin), which, when administered to humans,
provided them protection against smallpox, which Louis Pasteur and
others perpetuated. Jenner realized that milkmaids who had contact
with cowpox did not get smallpox. The process of distributing and
administrating vaccines is referred to as vaccination.

Vaccines can be prophylactic (e.g. to prevent or ameliorate the effects

of a future infection by any natural or "wild" pathogen), or therapeutic
(e.g. vaccines against cancer are also being investigated; see cancer
vaccine).

Vaccines may be dead or inactivated organisms or purified products

derived from them.

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There are four types of traditional vaccines:

• Vaccines containing killed microorganisms - these are previously

virulent micro-organisms that have been killed with chemicals or
heat. Examples are vaccines against flu, cholera, bubonic plague,
and hepatitis A.
• Vaccines containing live, attenuated microorganisms - these are
live micro-organisms that have been cultivated under conditions
that disable their virulent properties. They typically provoke
more durable immunological responses and are the preferred type
for healthy adults. Examples include yellow fever, measles,
rubella, and mumps.

• Toxoids - these are inactivated toxic compounds from micro-

organisms in cases where these (rather than the micro-organism
itself) cause illness. Examples of toxoid-based vaccines include
tetanus and diphtheria.

• Subunit - rather than introducing a whole inactivated or

attenuated micro-organism to an immune system, a fragment of it
can create an immune response. Characteristic examples include
the subunit vaccine against HBV that is composed of only the
surface proteins of the virus (produced in yeast) and the virus like
particle (VLP) vaccine against Human Papillomavirus (HPV) that
is composed of the viral major capsid protein.

The live tuberculosis vaccine is not the contagious strain, but a related
strain called "BCG"; it is used in the United States very infrequently.

A number of innovative vaccines are also in development and in use:

• Conjugate - certain bacteria have polysaccharide outer coats that

are poorly immunogenic. By linking these outer coats to proteins
(e.g. toxins), the immune system can be led to recognize the
polysaccharide as if it were a protein antigen. This approach is
used in the Haemophilus influenzae type B vaccine.

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 • Recombinant Vector - by combining the physiology of one
micro-organism and the DNA of the other, immunity can be
created against diseases that have complex infection processes
• DNA vaccination - in recent years a new type of vaccine, created
from an infectious agent's DNA called DNA vaccination, has
been developed. It works by insertion (and expression, triggering
immune system recognition) into human or animal cells, of viral
or bacterial DNA. Some cells of the immune system that
recognize the proteins expressed will mount an attack against
these proteins and cells expressing them. Because these cells live
for a very long time, if the pathogen that normally expresses
these proteins is encountered at a later time, they will be attacked
instantly by the immune system. One advantage of DNA vaccines
is that they are very easy to produce and store. As of 2006, DNA
vaccination is still experimental, but shows some promising
results.

Note that while most vaccines are created using inactivated or

attenuated compounds from micro-organisms, synthetic vaccines are
composed mainly or wholly of synthetic peptides, carbohydrates or
antigens.

A toxoid is a bacterial toxin whose toxicity has been weakened or

suppressed either by chemical (formalin) or heat treatment, while other
properties, typically immunogenicity, are maintained. Toxoids are used
in vaccines as they induce an immune response to the original toxin or
increase the response to another antigen. For example, the tetanus
toxoid is derived from the tetanospasmin produced by Clostridium
tetani and causing tetanus. The tetanus toxoid is used by many plasma
centers in the United States for the development of plasma rich
vaccines.

Antiserum (plural: antisera) is blood serum containing antibodies.

Antiserum is used to pass on passive immunity to many diseases.
Antiserum determines the antibody level or trail filtration by the
performance of taking blood sample from a laboratory animal. The
blood is allowed to clot, then the serum is removed for testing. Once
the antibody concentration reaches a desired level, the animal is bled.

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Immune serum obtained should contain antibodies produced in
response to the immunogenic stimulus. These antibodies are capable of
binding with the antigenic determinant (epitope) that had caused their
formation in some manner.

IV fluids

There are two types of fluids that are used for intravenous drips;
crystalloids and colloids. Crystalloids are aqueous solutions of mineral
salts or other water-soluble molecules. Colloids contain larger insoluble
molecules, such as gelatin; blood itself is a colloid.

The most commonly used crystalloid fluid is normal saline, a solution

of sodium chloride at 0.9% concentration, which is close to the
concentration in the blood (isotonic). Ringer's lactate or Ringer's
acetate (ASERING, patented brand name of Otsuka Indonesia) is
another isotonic solution often used for large-volume fluid replacement.
A solution of 5% dextrose in water, sometimes called D5W, is often
used instead if the patient is at risk for having low blood sugar or high
sodium. The choice of fluids may also depend on the chemical
properties of the medications being given.

Intravenous fluids must always be sterile

Antiseptics: Antiseptics (anti = against) are antimicrobial substances

that are applied to living tissue/skin to reduce the possibility of
infection, sepsis, or putrefaction. They should generally be
distinguished from antibiotics that destroy microorganisms within the
body, and from disinfectants, which destroy microorganisms found on
non-living objects. Some antiseptics are true germicides, capable of
destroying microbes (bactericidal), whilst others are bacteriostatic and
only prevent or inhibit their growth. Antibacterials are antiseptics that
only act against bacteria.

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Disinfectants are antimicrobial agents that are applied to non-living
objects to destroy microorganisms, the process of which is known as
disinfection. Disinfectants should generally be distinguished from
antibiotics that destroy microorganisms within the body, and from
antiseptics, which destroy microorganisms on living tissue. Sanitisers
are high level disinfectants that kill over 99.9% of a target
microorganism in applicable situations. Very few disinfectants and
sanitisers can sterilise (the complete elimination of all
microorganisms), and those that can depend entirely on their mode of
application. Bacterial endospores are most resistant to disinfectants;
however some viruses and bacteria also possess some tolerance.

Bioburden testing
The Bioburden is the population of viable micro-organisms present on
a material or product. In order to determine the bioburden requires the
removal of the micro-organisms from the material and then enumerate
them on a substrate media. There are two approaches. Repetitive
Recovery utilises the natural bioburden of the product. This
methodology determines the number of micro-organisms by several
washes, devolved on the percentage recovered in the first elution. This
method is suitable for products or materials that have a high bioburden.
The Inoculation Method, using an artificial bioburden of known
quantities, typically used to evaluate materials with low bioburden. The
organisms used in the validation are representative of the natural
bioburden in the product.

Sterility testing:

As part of sterilization validation, products and materials are subject to

sterility testing. This test uses techniques such as direct transfer to

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media, and membrane filtration, in order to detect the presence of
viable micro-organisms.

Microbial identification:

Isotron Laboratories perform bacterial identification, based on Gram

staining and biochemical testing. This is usually undertaken in
conjunction with bioburden and water testing, when they exceed action
or warning limits. Isotron works as a consultant with customers who
are investigating a contamination issue, and offers assistance in
identifying micro-organisms and thereby locating their source.

Other tests:

Our laboratory staff addresses our customers' microbiology needs, even

to the point of modifying tests to meet requirements. The wide range of
R&D projects undertaken by Isotron Laboratories includes microbial
limit testing, efficacy testing, microbial barrier testing, and age testing.
Our test facility enables our customers to take advantage of a
comprehensive range of product residual testing. Gas chromatographic
instrumentation is also available, testing by means of direct injection
and headspace analysis.

Pyrogen testing - LAL assay:

The FDA and US Pharmacopoeia Standards both state that medical

devices or pharmaceuticals which come into contact with re-circulating
blood, or the Central Nervous System (CNS), must not cause pyrogenic
reactions. Anything labelled 'pyrogen free' must use a recognised test
method to prove it. A pyrogen is a substance that, on introduction to
circulating blood cells or leached through the wall of the gut, raises
body temperature. Chemicals can cause this reaction. In the medical
device industry it is the endotoxins of gram-negative organisms that
usually effect the reaction. The water supply is the most likely source
of gram-negative bacteria, the outer cell walls of which include
endotoxins. Irradiation has no effect on them and they are present even

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after the cells die. Endotoxins are very difficult to remove, even by the
application of heat. The Pyrogen/LAL Assay facilitates a naturally
occurring reaction between endotoxins and an enzyme named Limulus
Amebocyte Lysate; an enzyme derived from the Horseshoe crab
(Limulus Polyphemus). The reaction between enzyme and endotoxins
results in a turbid clot. Isotron provides comprehensive test facilities for
the analysis of endotoxins on products and water.

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GLOSSARY:

1. Aerosol technically refers to airborne solid particles (also called

dust or particulate matter (PM)) or liquid droplets. In casual
language, aerosol refers to an aerosol spray can or the output of
such a can. The term aerosol derives from the fact that matter
"floating" in air is a suspension (a mixture in which solid or
liquid or combined solid-liquid particles are suspended in a
fluid). To differentiate suspensions from true solutions, the term
sol evolved—originally meant to cover dispersions of tiny (sub-
microscopic) particles in a liquid. With studies of dispersions in
air, the term aerosol evolved and now embraces liquid droplets,
solid particles, and combinations of these.
2. Disinfectants are antimicrobial agents that are applied to non-
living objects to destroy microorganisms, the process of which is
known as disinfection
3. Environmental Microbiology: The dynamic interactions of the
microbes with the physical and chemical make up of the world’s
many ecosystems are the subject of Environmental Microbiology.
4. Biota: Biota is the total collection of organisms of a geographic
region or a time period, from local geographic scales and
instantaneous temporal scales all the way up to whole-planet and
whole-timescale spatiotemporal scales. The biota of the Earth
lives in the biosphere
5. Ecology: Ecology (from Greek: oikos, "household"; and logos,
"knowledge") is the scientific study of the distribution and
abundance of living organisms and how the distribution and
abundance are affected by interactions between the organisms
and their environment. The environment of an organism includes
both physical properties, which can be described as the sum of
local abiotic factors such as insolation (sunlight), climate, and
geology, and biotic factors, which are other organisms that share
its habitat.
6. Ecosystem: An ecosystem is a natural unit consisting of all
plants, animals and micro organisms in an area functioning

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 together with all the non living physical factors of the
environment. In Short an Ecosystem is the total community of
organisms in a physically defined space
7. Environment: The natural surroundings around us in which we
live
8. Habitat: The physical space or location where a species lives
9. Biosphere: The biosphere (or sphere of life), sometimes
described as "the fourth envelope", is all living matter on the
planet or that portion of the planet occupied by life. It reaches
well into the other three spheres, although there are no permanent
inhabitants of the atmosphere. Relative to the volume of the
Earth, the biosphere is only the very thin surface layer which
extends from 11,000 meters below sea level to 15,000 meters
above.
10. Biotic: Biotic means relating to, produced by, or caused by
living organisms
11. Microbial Ecology: Microbial Ecology is the study of how
microorganisms interact with their biotic and abiotic
environments, with each other as well as with their neighbors and
hosts, to carry out their diverse functions.
12. BOD - Biochemical (or Biological) Oxygen Demand is a
chemical procedure for determining how fast biological
organisms use up oxygen in a body of water. It is used in water
quality management and assessment, Ecology and environmental
science. BOD is not an accurate quantitative test, although it
could be considered as an indication of the quality of a water
source.
13. COD: Chemical Oxygen Demand - In environmental
chemistry, the chemical oxygen demand (COD) test is
commonly used to indirectly measure the amount of organic
compounds in water. Most applications of COD determine the
amount of organic pollutants found in surface water (e.g. lakes
and rivers), making COD a useful measure of water quality. It is
expressed in milligrams per liter (mg/L), which indicates the
mass of oxygen consumed per liter of solution. Older references
may express the units as parts per million (ppm).

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14. Eutrophication refers to an increase in the primary
productivity of any ecosystem. It is caused by the increase of
chemical nutrients, typically compounds containing nitrogen or
phosphorus. It may occur on land or in water
15. Toxoids - these are inactivated toxic compounds from micro-
organisms in cases where these (rather than the micro-organism
itself) cause illness. Examples of toxoid-based vaccines include
tetanus and diphtheria
16. Pyrogen Testing: The FDA and US Pharmacopoeia Standards
both state that medical devices or pharmaceuticals which come
into contact with re-circulating blood, or the Central Nervous
System (CNS), must not cause pyrogenic reactions. Anything
labelled 'pyrogen free' must use a recognised test method to prove
it. A pyrogen is a substance that, on introduction to circulating
blood cells or leached through the wall of the gut, raises body
temperature.
17. Antiserum (plural: antisera) is blood serum containing
antibodies. Antiserum is used to pass on passive immunity to
many diseases
18. Vaccine: A vaccine is an antigenic preparation used to
establish immunity to a disease. The term derives from Edward
Jenner's use of cowpox ("vacca" means cow in Latin), which,
when administered to humans, provided them protection against
smallpox, which Louis Pasteur and others perpetuated. Jenner
realized that milkmaids who had contact with cowpox did not get
smallpox.
19. Vaccination: The process of distributing and administrating
vaccines is referred to as vaccination
20. Good Manufacturing Practice: Good Manufacturing Practice
or GMP (also referred to as 'cGMP' or 'current Good
Manufacturing Practice') is a term that is recognized worldwide
for the control and management of manufacturing and quality
control testing of foods and pharmaceutical products.
21. Pharmaceutical microbiology: Pharmaceutical Microbiology
is the part of industrial microbiology that is responsible for
creating medications. Pharmaceutical Microbiology is an applied
science discipline very relevant to infection and contamination

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 control. It draws upon understandings of mechanisms of
antimicrobial action and their relationship to resistance, hygiene
in the healthcare environment, and microbial pathogenicity and
behaviour.
22. Waterborne Diseases: Waterborne diseases are pathogenic
microorganisms which are directly transmitted when
contaminated drinking water is consumed. Contaminated
drinking water used in the preparation of food can be the source
of foodborne disease through consumption of the same
microorganisms.
23. MAV – Maximum Acceptable Values or
24. MAC – Maximum Acceptable Concentration
25. An estuary is a semi-enclosed coastal body of water with one
or more rivers or streams flowing into it, and with a free
connection to the open sea. Estuaries are often associated with
high rates of biological productivity.
26. Droplets: Droplets are usually formed by sneezing, coughing
or talking. Each consists of saliva and mucus. Droplets may also
contain hundreds of microorganisms which may be pathogenic if
discharged from diseased persons. Pathogens will be mostly of
respiratory tract origin. The size of the droplet determines the
time period during which they can remain suspended.
27. Droplet Nuclei: Small droplets in a warm, dry atmosphere tend
to evaporate rapidly and become droplet nuclei. Thus, the residue
of solid material left after drying up of a droplet is known as
droplet nuclei. These are small, 1-4µm, and light. They can
remain suspended in air for hours or days, traveling long
distances.
28. Infectious Dust - Large aerosol droplets settle out rapidly from
air on to various surfaces and get dried. Nasal and throat
discharges from a patient can also contaminate surfaces and
become dry. Disturbance of this dried material by bed making,
handling a handkerchief having dried secretions or sweeping
floors in the patient's room can generate dust particles which add
microorganisms to the circulating air.

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29. Nosocomial Infection: Infection acquired during the
hospitalization is called nosocomial infections. Infections,
manifested by the corresponding symptoms, after three days of
hospitalization can be regarded as nosocomial infection
according to Gleckman & Hibert, 1982 and Bonten&
Stobberingh, 1995.
30. Nosocomial Pathogens: The pathogens involved are
called as nosocomial pathogens.
31. Indoor Air:
32. Airborne Diseases: Diseases that are spread through air
containing pathogenic microorganisms which are directly
transmitted when inhaled and cause infections of the upper
respiratory tract (URT). The infections caused by such airborne
organisms tend to occur in epidemic form.
33. Epidemic form: It is the most infective form of organisms,
appearing infective in nature attacking large number of people
within a short time.
34. Montoux Test: It is the tuberculin test which is done by
intradermally injecting a purified protein derivative (PPD) taken
from culture filtrates of M.tuberculosis in a person, who tests
positive, a red, hardened area will appear at the site of infection
in about 48 hours. A positive reaction indicates that the person
either has an active case of tuberculosis or previously infected or
has been immunized. A positive chest film, the presence of acid-
fast bacteria in sputum or biopsy material and isolation and
speciation of mycobacterium confirm the diagnosis.
35. BCG: The Bacilli Calmette Guerin (BCG) is made with an
attenuated strain of mycobacterium and is administered to
persons. The BCG immunization is effective in preventing
childhood tuberculosis through its efficacy in preventing adult
TB is still in question.

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References:

1. Biochemical Engineering Fundamentals by J.E. Bailey and

P.F. Ollis, McGraw Hill Publication 2. . Principles of
fermentation technology by Stansbury, P.F., A. Whitaker
and S.J. Hall, 1997
2. Waste water Engineering, treatment & Disposal by Metcaff
& Reddy, Inc Publications. Tata Mac Craw Hill
3. Practical Medical Microbiology by Mackie McCartney
4. Microbiology by Anna K. Joshua
5. General Microbiology by Boyd
6. Text Book of Microbiology by R.Anantha Narayanan &
C.K. Jayaram Panicker
7. www.wikipedia.org
8. www.biologyofcancer.org

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