Environmental & Pharmaceutical Microbiology

Shanthi

Digitally signed by Shanthi DN: CN = Shanthi, C = IN, OU = Private Tutor - Health Science/ Basic Medical Sciences Date: 2009.04.14 11:03:01 +04'00'

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. 2. 3. 4. 5. 6. 7.

Nitrogen 78%, Oxygen 21 %, Argon 0.9%, Carbon dioxide 0.03%, Hydrogen 0.01 % and Other gases in trace amounts. 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. 2. 3. 4.

soil natural bodies of water plants 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. 9

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 10

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 11

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. 2. 3. 4.

atmospheric temperature, humidity, air current, The height at which the microorganisms are found etc. 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 12

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, ascospores 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. 13

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. 2. 3. 4. 5. 6.

Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, members of Enterobacteriaceae and Respiratory viruses. 14

Development of high antibiotic resistance is a potential problem among nosocomial pathogens. For example, Methicillin Resistant Staphylococcus aureus (MRSA) and gentamicin resistant Gramnegative 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.Bacterial

2.Viral

3.Fungal

1. 2. 3. 4. 5. 6. 7. 8.

Diphtheria, Septic sorethroat, Scarlet fever, Rheumatic fever. Tuberculosis, pneumonia, Meningitis, Whooping cough.

1. 2. 3. 4. 5. 6. 7. 8. 1.

Smallpox, Chickenpox, Measles, German Measles, Mumps, influenzea Common cold, Psittacosis. 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. 19

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. 20

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.

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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

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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.

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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).

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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 multilobulated 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.

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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.

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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.

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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.

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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.

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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:

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1. 2. 3. 4. 5. 6. 7.

Rhinoviruses – more than100 antigenic types Corona viruses Parainfluenza viruses Adenoviruses Respiratory syncytial viruses influenza viruses 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

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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.

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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.

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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.

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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.

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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, CrimeanCongo, 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

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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.

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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.

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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 nonsporulating 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

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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) 2) 3) 4) 5)

Sedimentation electrostatic attraction interception inertial impaction 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

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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

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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

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Class I Microbiological Safety Cabinet

Class II Microbiological Safety Cabinet

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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

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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.

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In large cities, domestic sewage is usually carried through a wellplanned 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

5. 6. 7. 8.

pollution of their winter feeding grounds Increased danger of swimming in the water diminished value of the water for the other recreational purposes Depletion of the oxygen supply of the water by unstable organic matter in sewage killing aquatic life. Creation of miscellaneous objectionable conditions such as offensive odours and accumulation of debris which decrease property values 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 ppm)

Water Quality

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.

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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

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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.

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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,

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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.

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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

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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 byproducts than chlorination. A disadvantage of ozone disinfection is the high cost of the ozone generation equipment and the requirements for highly skilled operators.

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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.

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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.

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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.

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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).

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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 87

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

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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.

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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

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Disease and Transmission

Amebiasis (handto-mouth)

Microbial Agent

Sources of Agent in Water Supply

Protozoan (Entamoeba Sewage, non-treated drinking histolytic) (Cyst-like water, flies in water supply appearance)

General Symptoms

Abdominal discomfort, fatigue, weight loss, diarrhea, gas pains Fever, abdominal pain, diarrhea

Cryptosporidiosis (oral)

Protozoan (Cryptosporidium parvum)

Collects on water filters and membranes that cannot be disinfected, animal manure, seasonal runoff of water.

Flu-like symptoms, watery diarrhea, loss of appetite, substantial loss of weight, bloating, increased gas, stomach

Cyclosporiasis

Protozoan parasite (Cyclospora cayetanensis)

Sewage, non-treated drinking water

cramps, nausea, vomiting, muscle aches, low-grade fever, and fatigue

Protozoan (Giardia lamblia) Most common intestinal parasite

Untreated water, poor disinfection, pipe breaks, leaks, groundwater contamination, campgrounds where humans and wildlife use same source of water. Beavers and muskrats act as a reservoir for Giardia.

Diarrhea, abdominal discomfort, bloating, gas and gas pains

Giardiasis (oralfecal) (hand-tomouth)

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Parasitic Infections Disease and Transmission

Microbial Agent

Sources of Agent in Water Supply

General Symptoms

Schistosomiasis (immersion)

Schistosoma

Contaminated fresh water with certain types of snails that carry schistosomes

Rash or itchy skin. Fever, chills, cough, and muscle aches

dracunculiasis

dracanculus medinensis

drinking water containing infective cyclops

allergic reaction,urticarial rash, nausea, vomiting, diarrhoea, asthmatic attack.

taeniasis solium

Taenia solium

contaminate drinking water with eggs

intestinal disturbances, neurologic manifestations, loss of weight, cysticercosis

fascioliasis

fasciola

GIT disturbance, diarrhea, liver contaminated drinking water with enlargement, cholangitis, cholecystitis, encysted metacercaria obstructive jaundice.

hymenolepiasis nana

hymenolepis nana

contaminated drinking water with mild GIT symptoms, nervous eggs manifestation

hyatidosis

echinococcus granulosus

hyatid cyst press on bile ductand blood contaminated drinking water with vessels, if it ruptured cause anaphylactic eggs shock.

coenurosis

multiceps multiceps

contaminated drinking water with inreases intacranial tension eggs

ascraiasis

ascaris lumbricoides

loefflers syndrome in lung, nausea, contaminated drinking water with vomiting, diarrhea, malnutrition, eggs underdevelopment,

enterobiasis

entrobius vermicularis

contaminated drinking water with peri-anal itch, nervous irritability, eggs hyperactivity and insomnia

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Disease

Morbility (cases per year)

1,500,000,000

Schistosomiasis

Mortality (deaths per year)

100,000

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.

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nutrient/ substance

Symbol/ formula

Normally found in fresh Health based water/surface guideline by the water/ground water WHO

Aluminium Ammonia

Al NH4

Antimony Arsenic Asbestos Barium Berillium Boron Cadmium Chloride Chromium Colour Copper Cyanide Dissolved oxygen Fluoride Hardness

Sb As

0,2 mg/l < 0,2 mg/l (up to 0,3 mg/l No guideline in anaerobic waters) < 4 µg/l 0.005 mg/l 0,01 mg/l No guideline 0,3 mg/l < 1 µg/l No guideline < 1 mg/l 0,3 mg/l < 1 µg/l 0,003 mg/l 250 mg/l < 2 µg/l 0,05 mg/l Not mentioned 2 mg/l 0,07 mg/l No guideline < 1,5 mg/l (up to 10) 1,5 mg/l No guideline

Hydrogen sulfide Iron Lead Manganese Mercury Molybdenum Nickel Nitrate and nitrite Turbidity pH Selenium Silver Sodium Sulfate Inorganic tin TDS Uranium Zinc

Ba Be B Cd Cl Cr+3, Cr+6 Cu CNO2 F mg/l CaCO3 H2 S Fe Pb Mn Hg Mb Ni NO3, NO2

Se Ag Na SO4 Sn

0,5 - 50 mg/l

< 0,5 µg/l < 0,01 mg/l < 0,02 mg/l

< < 0,01 mg/l 5 – 50 µg/l < 20 mg/l

U Zn

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No guideline No guideline 0,01 mg/l 0,5 mg/l 0,001 mg/l 0,07 mg/l 0,02 mg/l 50 mg/l total nitrogen Not mentioned No guideline 0,01 mg/l No guideline 200 mg/l 500 mg/l No guideline No guideline 1,4 mg/l 3 mg/l

Organic compounds Group

Substance

Formula

Chlorinated alkanes Carbon tetrachloride Dichloromethane 1,1-Dichloroethane 1,2-Dichloroethane 1,1,1-Trichloroethane Chlorinated ethenes 1,1-Dichloroethene 1,2-Dichloroethene Trichloroethene Tetrachloroethene Aromatic Benzene hydrocarbons Toluene Xylenes Ethylbenzene Styrene Polynuclear Aromatic Hydrocarbons (PAHs) Chlorinated Monochlorobenzene (MCB) benzenes Dichlorobenzenes 1,2-Dichlorobenzene (DCBs) (1,2-DCB) 1,3-Dichlorobenzene (1,3-DCB) 1,4-Dichlorobenzene (1,4-DCB) Trichlorobenzenes (TCBs) Miscellaneous Di(2-ethylhexyl)adipate (DEHA) organic constituents Di(2-ethylhexyl)phthalate (DEHP) Acrylamide Epichlorohydrin (ECH) Hexachlorobutadiene (HCBD) Ethylenediaminetetraacetic acid (EDTA) Nitrilotriacetic acid (NTA) Organotins Dialkyltins Tributil oxide (TBTO)

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C Cl4 C H2 Cl2 C2 H4 Cl2 Cl CH2 CH2 Cl CH3 C Cl3 C2 H2 Cl2 C2 H2 Cl2 C2 H Cl3 C2 Cl4 C6 H6 C7 H8 C8 H10 C8 H10 C8 H8 C2 H3 N1 O5 P1 3 C6 H5 Cl C6 H4 Cl2

Health based guideline by the WHO 2 µg/l 20 µg/l No guideline 30 µg/l 2000 µg/l 30 µg/l 50 µg/l 70 µg/l 40 µg/l 10 µg/l 700 µg/l 500 µg/l 300 µg/l 20 µg/l 0.7 µg/l 300 µg/l 1000 µg/l

C6 H4 Cl2

No guideline

C6 H4 Cl2

300 µg/l

C6 H3 Cl3 C22 H42 O4 C24 H38 O4 C3 H5 N O C3 H5 Cl O C4 Cl6 C10 H12 N2 O8 N(CH2COOH)3 R2 Sn X2 C24 H54 O Sn2

20 µg/l 80 µg/l 8 µg/l 0.5 µg/l 0.4 µg/l 0.6 µg/l 200 µg/l 200 µg/l No guideline 2 µg/l

Pesticides Substance

Formula

Alachlor Aldicarb Aldrin and dieldrin

C14 H20 Cl N O2 C7 H14 N2 O4 S C12 H8 Cl6/

Health based guideline by the WHO 20 µg/l 10 µg/l 0.03 µg/l

Atrazine Bentazone Carbofuran Chlordane Chlorotoluron DDT 1,2-Dibromo-3-chloropropane 2,4-Dichlorophenoxyacetic acid (2,4-D) 1,2-Dichloropropane 1,3-Dichloropropane 1,3-Dichloropropene Ethylene dibromide (EDB) Heptachlor and heptachlor epoxide Hexachlorobenzene (HCB) Isoproturon Lindane MCPA Methoxychlor Metolachlor Molinate Pendimethalin Pentachlorophenol (PCP) Permethrin Propanil Pyridate Simazine Trifluralin Chlorophenoxy 2,4-DB herbicides (excluding Dichlorprop 2,4-D and MCPA) Fenoprop MCPB Mecoprop 2,4,5-T

C12 H8 Cl6 O C8 H14 Cl N5 C10 H12 N2 O3 S C12 H15 N O3 C10 H6 Cl8 C10 H13 Cl N2 O C14 H9 Cl5 C3 H5 Br2 Cl C8 H6 Cl2 O3 C3 H6 Cl2 C3 H6 Cl2 CH3 CHClCH2 Cl Br CH2 CH2 Br C10 H5 Cl7 C10 H5 Cl7 O C12 H18 N2 O C6 H6 Cl6 C9 H9 Cl O3 (C6H4OCH3)2CHCCl3 C15 H22 Cl N O2 C9 H17 N O S C13 H19 O4 N3 C6 H Cl5 O C21 H20 Cl2 O3 C9 H9 Cl2 N O C19H23ClN2O2S C7 H12 Cl N5 C13 H16 F3 N3 O4 C10 H10 Cl2 O3 C9 H8 Cl2 03 C9H7Cl3O3 C11 H13 Cl O3 C10H11ClO3 C8 H5 Cl3 O3

2 µg/l 30 µg/l 5 µg/l 0.2 µg/l 30 µg/l 2 µg/l 1 µg/l 30 µg/l No guideline 20 µg/l No guideline No guideline 0.03 µg/l 1 µg/l 9 µg/l 2 µg/l 2 µg/l 20 µg/l 10 µg/l 6 µg/l 20 µg/l 9 µg/l 20 µg/l 20 µg/l 100 µg/l 2 µg/l 20 µg/l 90 µg/l 100 µg/l 9 µg/l No guideline 10 µg/l 9 µg/l

99

Disinfectants and disinfectant by-products Group

Substance

Disinfectants

NHnCl(3-n), where n = 0, 1 or 2 Chlorine Cl2 Chlorine dioxide ClO2 Iodine I2 Bromate Br O3Chlorate Cl O3Chlorite Cl O2Chlorophenols 2-Chlorophenol (2-CP) C6 H5 Cl O 2,4-Dichlorophenol (2,4-DCP) C6 H4 Cl2 O 2,4,6-Trichlorophenol (2,4,6-TCP) C6 H3 Cl3 O Formaldehyde HCHO MX (3-Chloro-4-dichloromethyl-5-hydroxy-2(5H)C5 H3 Cl3 O3 furanone) Trihalomethanes Bromoform C H Br3 Dibromochloromethane CH Br2 Cl Bromodichloromethane CH Br Cl2 Chloroform CH Cl3 Chlorinated acetic acids Monochloroacetic acid C2 H3 Cl O2 Dichloroacetic acid C2 H2 Cl2 O2 Trichloroacetic acid C2 H Cl3 O2 Chloral hydrate (trichloroacetaldehyde) C Cl3 CH(OH)2 Chloroacetones C3 H5 O Cl Halogenated acetonitriles Dichloroacetonitrile C2 H Cl2 N Dibromoacetonitrile C2 H Br2 N Bromochloroacetonitrile CH Cl2 CN Trichloroacetonitrile C2 Cl3 N Cyanogen chloride Cl CN Chloropicrin C Cl3 NO2

Disinfectant byproducts

Formula

Chloramines

100

Health based guideline by the WHO 3 mg/l

5 mg/l No guideline No guideline 25 µg/l No guideline 200 µg/l No guideline No guideline 200 µg/l 900 µg/l No guideline 100 µg/l 100 µg/l 60 µg/l 200 µg/l No guideline 50 µg/l 100 µg/l 10 µg/l No guideline 90 µg/l 100 µg/l No guideline 1 µg/l 70 µg/l No guideline

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

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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.

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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 recontamination 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 morevisible 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

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2.

3.

4. 5. 6.

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. 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. 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. Rivers, canals and low land reservoirs: Low land surface waters will have a significant bacterial load and may also contain algae, suspended solids and a variety of dissolved constituents. Atmospheric water generation is a new technology that can provide high quality drinking water by extracting water from the air by cooling the air and thus condensing water vapour. 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

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2.

3.

4. 5.

constructed so that accidental contamination does not occur. 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. 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. 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. 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.

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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

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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. 110

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 microorganisms 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. 111

(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 112

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 113

2.

3.

4.

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7.

new pathogens. 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. 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. 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. 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. 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. The use of iron in removing arsenic from water. See Arsenic contamination of groundwater Water purification solutions 114

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 115

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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2. Correct Choice of Cleaning Equipment Regularly monitored

1. Equipments of good design and properly maintained

4. Manufacturing Premises of Good Design & Regularly Monitored

3. Quality control of raw

material

5. Quality control of

finished product

6. Written procedures &

7. Stained staff wearing

other documentation

protective clothing

Quality control of packaging 8.

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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. 118

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 interrelated. 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. 120

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 121

• 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. 123

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 124

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, xrays or certain chemicals. Selection and further reproduction of the higher yielding strains over many generations can raise yields by 20fold 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

Ammonia

Pure Urea

Nitrate

Nitrate salts

Phosphorus source

Phosphate salts

ammonia

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

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or

ammonium

salts

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 microorganisms 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. 131

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 133

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 (submicroscopic) 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 nonliving 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 microorganisms 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 138

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 acidfast 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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