Lec 21-22 Water Quality PDF

WATER QUALITY PARAMETERS ENVIRONMENTAL BIOTECHNOLOGY WATER QUALITY The most popular definition of water quality is “it is the physical, chemical, and biological characteristics of water”. Water quality is a measure of the condition of water relative to the requirements of one or more bioti...

WATER QUALITY PARAMETERS ENVIRONMENTAL BIOTECHNOLOGY WATER QUALITY The most popular definition of water quality is “it is the physical, chemical, and biological characteristics of water”. Water quality is a measure of the condition of water relative to the requirements of one or more biotic species and/or to any human need or purpose. For example, water that is clean enough for swimming and fishing may not be clean enough for drinking and cooking. Water quality standards (limits on the amount of impurities allowed in water intended for a particular use) provide a legal framework for the prevention of water pollution of all types. Water quality classification Water quality can be classified into four types—potable water, palatable water, contaminated (polluted) water, and infected water. 1. Potable water: It is safe to drink, pleasant to taste, and usable for domestic purposes. 2. Palatable water: It is esthetically pleasing; it considers the presence of chemicals that do not cause a threat to human health. 3. Contaminated (polluted) water: It is that water containing unwanted physical, chemical, biological, or radiological substances, and it is unfit for drinking or domestic use. 4. Infected water: It is contaminated with pathogenic organism. Water quality standards Stream standards are those that classify streams, rivers, and lakes on the basis of their maximum beneficial use; they set allowable levels of specific substances or qualities (e.g., dissolved oxygen, turbidity, pH) allowed in those bodies of water, based on their given classification. Effluent (water outflow) standards set specific limits on the levels of contaminants (e.g., biochemical oxygen demand, suspended solids, nitrogen) allowed in the final discharges from wastewater- treatment plants. Drinking-water standards include limits on the levels of specific contaminants allowed in potable water delivered to homes for domestic use. In India Bureau of Indian Standards has recommended water quality parameters for different uses in the standard IS2296:1992. Water Quality Standards in India(Source IS 2296:1992). For drinking water- BIS-Drinking Water Specifications (IS:10500-2012) Water quality parameters ❑Physical, Chemical and microbiological properties. ❑Water quality is determined according to purpose of use (drinking, agriculture or industrial) ❑Water used for certain purpose is compared with standards for that type of water ❑Standards put into account not to affect negatively public health, plant growth, or industrial processes  Types of water quality parameters Physical parameters Chemical parameters Biological parameters 1 Turbidity pH Bacteria 2 Temperature Acidity Algae 3 Color Alkalinity Viruses 4 Taste and odor Chloride Protozoa 5 Solids Chlorine residual 6 Electrical conductivity (EC) Sulfate 7 Nitrogen 8 Fluoride 9 Iron and manganese 10 Copper and zinc 11 Hardness 12 Dissolved oxygen 13 Biochemical oxygen demand (BOD) 14 Chemical oxygen demand (COD) 15 Toxic inorganic substances 16 Toxic organic substances 1. General physical parameters: 1. Turbidity (JTU, FTU, NTU, ppm, SiO2 ) 2. Temperature 3. Color (g.pt/m3 ) 4. Odor and taste 5. Conductivity (µs/cm or µs/m) 6. Solids content (mg/l or g/m3 )  Turbidity: is related to the presence of finely suspended particles of inorganic or organic origin. Turbidity It is the capacity of a water for absorbing or scattering light and is measured by the concentration of a standard like fine silica or formazine that produces an equivalent effect. Measurements of turbidity do not give complete information about the size, number, mass or type of particles that scatter or absorb light.  Turbidity is measured by an instrument called nephelometric turbidimeter, which expresses turbidity in terms of NTU or TU. A TU is equivalent to 1 mg/L of silica in suspension. Jackson Turbidity Units (JTU): Turbidity unit based upon the visibility of the flame of a candle through the water. FTU: Standard formazine, which gives a reproducible turbidity to water. Nephlometer Turbidity Units (NTU): Different standards are used. Turbidity more than 5 NTU can be visible to the average person while Turbidity in muddy water exceeds 100 NTU. Groundwater normally has very low turbidity because of the natural filtration that occurs as the water penetrates through the soil Turbidimeters used at a water purification plant to measure turbidity (in NTU) of raw water and clear water after filtration  Temperature: Temperature: is an important parameter because many physical, chemical and biological processes, which can occur in water are temperature –dependent. Water temperature and water density are directly related. As the temperature of water increases or decreases, it will alter the density of water. Palatability, viscosity, solubility, odors, and chemical reactions are influenced by temperature. Thereby, the sedimentation and chlorination processes and biological oxygen demand (BOD) are temperature dependent. It also affects the biosorption process of the dissolved heavy metals in water. Most people find water at temperatures of 10–15°C most palatable Color: Color: is due to the presence of natural organic matter (humic substance giving the yellow color. The color may also by caused by certain industrial waste and by some metallic complexes. Color is measured by comparing the water sample with standard color solutions or colored glass disks. Color is measured by the concentration of standard Platinum – Cobalt solution that produces an equivalent effect and expressed in (g.pt/m3 ) EPA Secondary Drinking Water Recommendation is for color of less than 15 Platinum Cobalt Units (PCU) 1 unit - the color of distilled water containing 1 milligram of platinum as potassium chloroplatinate per liter  The color of a water sample can be reported as follows: Apparent color is the entire water sample color and consists of both dissolved and suspended components color. True color is measured after filtering the water sample to remove all suspended material. Color is graded on scale of 0 (clear) to 70 color units. Pure water is colorless, which is equivalent to 0 color units. Color is reduced or removed from water through the use of coagulation, settling and filtration techniques.  Odor and Taste: Odor and Taste: is often due to dissolved organic impurities, such as phenols, chlorophenols, sewage component chemicals like iron, manganese, zinc etc. The numerical value of odor or taste is determined quantitatively by measuring a volume of sample A and diluting it with a volume of sample B of an odor-free distilled water so that the odor of the resulting mixture is just detectable at a total mixture volume of 200 ml. The unit of odor or taste is expressed in terms of a threshold number as follows: TON or TTN=(A+B)/A where TON is the Threshold odor number and TTN is the Threshold taste number. Algae can produce severe taste and odor problems Conductivity: Conductivity: a measure of the ability of water to pass an electrical current. This ability is directly dependent on the concentration of conductive ions present in the water. These conductive ions are originated due to inorganic materials such as chlorides, alkalis, carbonate and sulfides compounds and dissolved salts. Most inorganic acids, bases and salts are good conductors The standard unit of electrical resistance is the ohm, the standard unit of electrical conductance is its inverse, the mho or recently Siemen. Pure water is said to be a bad conductor of electricity. Normal water is said to have impurities present in the form of ions called minerals etc. These ions are known to be responsible for the conduction of electric current in the water. Conductivity of Water Units: The conductivity of water can be measured using multiple units. Some of them are: Units SI units Siemens per meter [S/m] Low Conductivity (0 to 200 µS/cm) Types of watermillimhos per centimeter U.S units Conductivity Value [mmho/cm] is an indicator of pristine conditions. Pure distilled and Mid range conductivity (200 to 0.05 µS/cm Deionized water 1000 µS/cm) is the normal Seawater 50 mS/cm background for most major rivers. Because dissolved salts and other Drinking water 200 to 800 µS/cm. inorganic chemicals conduct Rain or Snow water 2 to 100 µS/cm electrical current, conductivity increases as salinity increases. Solid component Solids occur in water either in solution or in suspension. These two types of solids can be identified by using a glass fiber filter that the water sample passes through. By definition, the suspended solids are retained on the top of the filter as total suspended solids (TSS)and the dissolved solids pass through the filter with the water. If the filtered portion of the water sample is placed in a small dish and then evaporated, the solids remain as a residue. This material is usually called total dissolved solids or TDS. Total solid(TS)=Total dissolved solid(TDS)+Total suspended solid(TSS)  The residue of TSS and TDS after heating to dryness for a defined period of time and at a specific temperature is defined as fixed solids. Volatile solids are those solids lost on ignition (heating to 550°C) Solid component Total Solids (TS): The total of all solids in a water sample Total Suspended Solids (TSS): The amount of filterable solids in a water sample, filters are dried and weighed Total Dissolved Solids (TDS): Non-filterable solids that pass through a filter with a pore size of 2.0 micron, after filtration the liquid is dried and residue is weighed EPA Secondary Drinking Water Recommendation is for TDS of less than 500mg/L Volatile Solids (VS): Volatile solids are those solids lost on heating to 550 degrees C - rough approximation of the amount of organic matter present in the solid fraction of wastewater  Water can be classified by the amount of TDS per liter as follows Freshwater: 5000 mg/L Chemical parameters of water quality 1. pH 1. Iron and Manganese 2. Acidity 2. Copper and Zinc 3. Alkalinity 3. Hardness 4. Chloride 4. BOD 5. Chlorine Residual 5. COD 6. Sulfate 6. Toxic inorganic substances 7. Nitrogen 7. Toxic organic substances 8. Fluoride 8. Radioactive substances pH- Acidity or Alkalinity It is defined as the negative logarithm of the hydrogen ion concentration. It is a dimensionless number indicating the strength of an acidic or a basic solution. Actually, pH of water is a measure of how acidic/basic water is.  Pure water is neutral, with a pH close to 7.0 at 25°C. Normal rainfall has a pH of approximately 5.6 (slightly acidic) owing to atmospheric carbon dioxide gas. Safe ranges of pH for drinking water are from 6.5 to 8.5 for domestic use. A high pH makes the taste bitter and decreases the effectiveness of the chlorine disinfection, thereby causing the need for additional chlorine. The amount of oxygen in water increases as pH rises. Low-pH water will corrode or dissolve metals and other substances. The effects of pH on animals and plants Even moderately acidic water (low pH) can decrease the number of hatched fish eggs, irritate fish and aquatic insect gills, and damage membranes. Water with very low or high pH is fatal. A pH below 4 or above 10 will kill most fish, and very few animals can endure water with a pH below 3 or above 11. Amphibians are extremely endangered by low pH because their skin is very sensitive to contaminants. Some scientists believe that the current decrease in amphibian population throughout the globe may be due to low pH levels induced by acid rain. The effects of pH on other chemicals in water Heavy metals such as cadmium, lead, and chromium dissolve more easily in highly acidic water (lower pH). This is important because many heavy metals become much more toxic when dissolved in water. A change in the pH can change the forms of some chemicals in the water. Therefore, it may affect aquatic plants and animals. For instance, ammonia is relatively harmless to fish in neutral or acidic water. However, as the water becomes more alkaline (the pH increases), ammonia becomes progressively more poisonous to these same organisms. Acidity Acidity in water is usually due to carbon dioxide, mineral acids, and hydrolyzed salts such as ferric and aluminum sulfates. Acids can influence many processes such as corrosion, chemical reactions and biological activities. Carbon dioxide from the atmosphere or from the respiration of aquatic organisms causes acidity when dissolved in water by forming carbonic acid (H2CO3). The level of acidity is determined by titration with standard sodium hydroxide (0.02 N) using phenolphthalein as an indicator. Alkalinity The alkalinity of water is its acid-neutralizing capacity comprised of the total of all titratable bases. The measurement of alkalinity of water is necessary to determine the amount of lime and soda needed for water softening (e.g., for corrosion control in conditioning the boiler feed water). Alkalinity of water is mainly caused by the presence of hydroxide ions (OH−), bicarbonate ions (HCO3−), and carbonate ions (CO32−), or a mixture of two of these ions in water. Alkalinity is determined by titration with a standard acid solution (H2SO4 of 0.02 N) using selective indicators (methyl orange or phenolphthalein). Chloride Chloride occurs naturally in groundwater, streams, and lakes, but the presence of relatively high chloride concentration in freshwater (about 250 mg/L or more) may indicate wastewater pollution. Chlorides may enter surface water from several sources including chloride-containing rock, agricultural runoff, and wastewater. Chloride ions (Cl− ) in drinking water do not cause any harmful effects on public health, but high concentrations can cause an unpleasant salty taste for most people. The chloride concentration in water, is measured by the titration method by silver nitrate. Sulfate Sulfate ions (SO42−) occur in natural water and in wastewater. The high concentration of sulfate in natural water is usually caused by leaching of natural deposits of sodium sulfate (Glauber’s salt) or magnesium sulfate (Epson salt). If high concentrations are consumed in drinking water, there may be objectionable tastes or unwanted laxative effects, but there is no significant danger to public health. Nitrogen There are four forms of nitrogen in water and wastewater: organic nitrogen, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. If water is contaminated with sewage, most of the nitrogen is in the forms of organic and ammonia, which are transformed by microbes to form nitrites and nitrates. A high concentration of nitrate in surface water can cause eutrophication. Nitrates can enter the groundwater from chemical fertilizers used in the agricultural areas. Excessive nitrate concentration (more than 10 mg/L) in drinking water causes an immediate and severe health threat to infants. The nitrate ions react with blood hemoglobin, thereby reducing the blood’s ability to hold oxygen which leads to a disease called blue baby syndrome or methemoglobinemia. Fluoride A moderate amount of fluoride ions (F−) in drinking water contributes to good dental health. About 1.0 mg/L is effective in preventing tooth decay, particularly in children. Excessive amounts of fluoride cause discolored teeth, a condition known as dental fluorosis. The maximum allowable levels of fluoride in public water supplies ranges from 1.4 mg/L; in colder climates, up to 2.4 mg/L in warmer areas. Iron and manganese Although iron (Fe) and manganese (Mn) do not cause health problems, they impart a noticeable bitter taste to drinking water even at very low concentration. These metals usually occur in groundwater in solution as ferrous (Fe2+) and manganous (Mn2+) ions. When these ions are exposed to air, they form the insoluble ferric (Fe3+) and manganic (Mn3+) forms making the water turbid and unacceptable to most people. These ions can also cause black or brown stains on laundry and plumbing fixtures. They are measured by many instrumental methods such as atomic absorption spectrometry. Copper (Cu) and zinc (Zn) Copper (Cu) and zinc (Zn) are nontoxic if found in small concentrations. Actually, they are both essential and beneficial for human health and growth of plants and animals. They can cause undesirable tastes in drinking water. At high concentrations, zinc imparts a milky appearance to the water. They are measured by the same methods used for iron and manganese measurements. Hardness Hardness is a term used to express the properties of highly mineralized waters. The dissolved minerals in water cause problems such as scale deposits in hot water pipes and difficulty in producing lather with soap. Calcium (Ca2+) and magnesium (Mg2+) ions cause the greatest portion of hardness in naturally occurring waters. They enter water mainly from contact with soil and rock, particularly limestone deposits. These ions are present as bicarbonates, sulfates, and sometimes as chlorides and nitrates.  Generally, groundwater is harder than surface water. There are two types of hardness: Temporary hardness which is due to carbonates and bicarbonates can be removed by boiling, and Permanent hardness which is remaining after boiling is caused mainly by sulfates and chlorides. From health viewpoint, hardness up to 500 mg/L is safe, but more than that may cause a laxative effect. Water classification Total hardness concentration as mg/L as CaCO3 Soft water 300 mg/L as CaCO3 Dissolved oxygen Dissolved oxygen (DO) is considered to be one of the most important parameters of water quality in streams, rivers, and lakes. It is a key test of water pollution. The higher the concentration of dissolved oxygen, the better the water quality. Oxygen is slightly soluble in water and very sensitive to temperature. For example, the saturation concentration at 20°C is about 9 mg/L and at 0°C is 14.6 mg/L. There are three main methods used for measuring dissolved oxygen concentrations: the colorimetric method—quick and inexpensive, the Winkler titration method—traditional method, and the electrometric method Biochemical oxygen demand (BOD) Bacteria and other microorganisms use organic substances for food. As they metabolize organic material, they consume oxygen. The organics are broken down into simpler compounds, such as CO2 and H2O, and the microbes use the energy released for growth and reproduction. When this process occurs in water, the oxygen consumed is the DO in the water. If oxygen is not continuously replaced by natural or artificial means in the water, the DO concentration will reduce as the microbes decompose the organic materials. This need for oxygen is called the biochemical oxygen demand (BOD). The more organic material there is in the water, the higher the BOD used by the microbes will be. BOD is used as a measure of the power of sewage; strong sewage has a high BOD and weak sewage has low BOD Measurement of BOD The complete decomposition of organic material by microorganisms takes time, usually 20 d or more under ordinary circumstances. The quantity of oxygen used in a specified volume of water to fully decompose or stabilize all biodegradable organic substances is called the ultimate BOD or BODL. BOD is a function of time. At time = 0, no oxygen will have been consumed and the BOD = 0. As each day goes by, oxygen is used by the microbes and the BOD increases. Ultimately, the BODL is reached and the organic materials are completely decomposed. Chemical oxygen demand (COD) The chemical oxygen demand (COD) is a parameter that measures all organics: the biodegradable and the non-biodegradable substances. It is a chemical test using strong oxidizing chemicals (potassium dichromate), sulfuric acid, and heat, and the result can be available in just 2 h. COD values are always higher than BOD values for the same sample. Toxic inorganic substances A wide variety of inorganic toxic substances may be found in water in very small or trace amounts. Even in trace amounts, they can be a danger to public health. Some toxic substances occur from natural sources but many others occur due to industrial activities and/or improper management of hazardous waste. They can be divided into two groups: Metallic compounds: Non –metallic compounds: Metallic compounds: and Non-Metallic compounds: Metallic compounds: This group includes some heavy metals that are toxic, namely, cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), silver (Ag), arsenic (As), barium (Ba), thallium (Tl), and selenium (Se). They have a wide range of dangerous effects that differ from one metal to another. They may be acute fatal poisons such as (As) and (Cr6+) or may produce chronic diseases such as (Cd, Hg, Pb, and Tl). The heavy metals concentration can be determined by atomic absorption photometers, spectrophotometer, or inductively coupled plasma (ICP) for very low concentration. Metallic compounds: and Non-Metallic compounds: Nonmetallic compounds: This group includes nitrates (NO3−) and cyanides (CN−), Regarding cyanide, it causes oxygen deprivation by binding the hemoglobin sites and prevents the red blood cell from carrying the oxygen. This causes a blue skin color syndrome, which is called cyanosis. It also causes chronic effects on the central nervous system and thyroid. Cyanide is normally measured by colorimetric, titrimetric, or electrometric methods Toxic organic substances There are more than 100 compounds in water that have been listed in the literature as toxic organic compounds. They will not be found naturally in water; they are usually man-made pollutants. These compounds include insecticides, pesticides, solvents, detergents, and disinfectants. They are measured by highly sophisticated instrumental methods, namely, gas chromatographic (GC), high-performance liquid chromatographic (HPLC), and mass spectrophotometric methods. Radioactive substances Potential sources of radioactive substances in water include wastes from nuclear power plants, industries, or medical research using radioactive chemicals and mining of uranium ores or other radioactive materials. When radioactive substances decay, they release beta, alpha, and gamma radiation. Exposure of humans and other living things to radiation can cause genetic and somatic damage to the living tissues. Radon gas is of a great health concern because it occurs naturally in groundwater and is a highly volatile gas, which can be inhaled during the showering process.. Table of Secondary Standards Contaminant Secondary MCL Noticeable Effects above the Secondary MCL Aluminum 0.05 to 0.2 mg/L* colored water Chloride 250 mg/L salty taste Color 15 color units visible tint Copper 1.0 mg/L metallic taste; blue-green staining Corrosivity Non-corrosive metallic taste; corroded pipes/ fixtures staining Fluoride 2.0 mg/L tooth discoloration Foaming agents 0.5 mg/L frothy, cloudy; bitter taste; odor Iron 0.3 mg/L rusty color; sediment; metallic taste; reddish or orange staining Manganese 0.05 mg/L black to brown color; black staining; bitter metallic taste 3 TON (threshold odor Odor "rotten-egg", musty or chemical smell number) low pH: bitter metallic taste; corrosion pH 6.5 - 8.5 high pH: slippery feel; soda taste; deposits Silver 0.1 mg/L skin discoloration; graying of the white part of the eye Sulfate 250 mg/L salty taste Total Dissolved Solids 500 mg/L hardness; deposits; colored water; staining; salty taste (TDS) Zinc 5 mg/L metallic taste Biological Parameters of water quality Bacteria Algae Viruses Protozoa Indicator organisms  Biological Parameters of water quality One of the most helpful indicators of water quality may be the presence or lack of living organisms. Biologists can survey fish and insect life of natural waters and assess the water quality on the basis of a computed species diversity index (SDI); hence, a water body with a large number of well-balanced species is regarded as a healthy system. Some organisms can be used as an indication for the existence of pollutants based on their known tolerance for a specified pollutant. Bacteria Types: Spheroid, rod curved rod, spiral, filamentous. Bacteria that require oxygen for their metabolism are called aerobic bacteria, while those live only in an oxygen-free environment are called anaerobic bacteria. Some species called facultative bacteria can live in either the absence or the presence of oxygen Some important bacteria in waste water treatment Pseudomonas:- reduce NO3 to N2 , So it is very important in biological nitrate removal in treatment works. Zoogloea:- helps through its slime production in the formation of flocs in the aeration tanks. Sphaerotilus natuns: Causes sludge bulking in the aeration tanks. Bdellovibrio: destroy pathogens in biological treatment. Acinetobacter: Store large amounts of phosphate under aerobic conditions and release it under an – anaerobic condition so, they are useful in phosphate removal. Nitrosomonas: transform NH4 into NO2 - Nitrobacter: transform NO2 - to NO3 - Coliform bacteria:- The most common type is E-Coli or Echerichia Coli, (indicator for the presence of pathogens). E-Coli is measured in (No/100mL  At low temperatures, bacteria grow and reproduce slowly. As the temperature increases, the rate of growth and reproduction doubles in every additional 10°C (up to the optimum temperature for the species). The majority of the species of bacteria having an optimal temperature of about 35°C. A lot of dangerous waterborne diseases are caused by bacteria, namely, typhoid and paratyphoid fever, leptospirosis, tularemia, shigellosis, and cholera. Sometimes, the absence of good sanitary practices results in gastroenteritis outbreaks of one or more of those diseases  Algae Algae are microscopic plants, which contain photosynthetic pigments, such as chlorophyll. They are also important for wastewater treatment in stabilization ponds. Algae are primarily nuisance organisms in the water supply because of the taste and odor problems they create. Certain species of algae cause serious environmental and public health problems; for example, blue-green algae can kill cattle and other domestic animals if the animals drink water containing those species. Cause eutrophication phenomena. (negative effect) Cause taste and problems when decayed. (negative effect) Viruses Viruses are the smallest biological structures known to contain all genetic information necessary for their own reproduction. They can only be seen by a powerful electronic microscope. Viruses are parasites that need a host to live. They can pass through filters that do not permit the passage of bacteria. Waterborne viral pathogens are known to cause infectious hepatitis and poliomyelitis. Most of the waterborne viruses can be deactivated by the disinfection process conducted in the water treatment plant  Protozoa Protozoa are single-celled microscopic animal, consume solid organic particles, bacteria, and algae for food, and they are in turn ingested as food by higher level multicellular animals. Aquatic protozoa are floating freely in water and sometimes called zooplankton. They form cysts that are difficult to inactivate by disinfection Indicator organisms of water pollution A very important biological indicator of water and pollution is the group of bacteria called coliforms. Pathogenic coliforms always exist in the intestinal system of humans, and millions are excreted with body wastes. Consequently, water that has been recently contaminated with sewage will always contain coliforms. A particular species of coliforms found in domestic sewage is Escherichia coli or E. coli. Even if the water is only slightly polluted, they are very likely to be found. There are roughly 3 million of E. coli bacteria in 100 mL volume of untreated sewage. Coliform bacteria are aggressive organisms and survive in the water longer than most pathogens. COLIFORM TEST There are normally two methods to test the coliform bacteria— 1. Membrane filter method and 2. Multiple-tube fermentation method OR Most probable number technique  Testing for coliforms: membrane filter method A measured volume of sample is filtered through a special membrane filter by applying a partial vacuum. The filter, a flat paper-like disk, has uniform microscopic pores small enough to retain the bacteria on its surface while allowing the water to pass through. The filter paper is then placed in a petri dish, which contains a special culture medium that the bacteria use as a food source. Then, the petri dish is usually placed in an incubator, which keeps the temperature at 35°C, for 24 h. After incubation, colonies of coliform bacteria each containing millions of organisms will be visible. The coliform concentration is obtained by counting the number of colonies on the filter; each colony counted represents only one coliform in the original sample Most Probable Number Technique or Multiple fermentation tube technique Multiple fermentation tube technique or MPN technique It is the only procedure that can be used if water samples are very turbid or if semi-solids such as sediments or sludges are to be analysed. The procedure followed is fundamental to bacteriological analyses and the test is used in many countries. It is customary to report the results of the multiple fermentation tube test for coliforms as a most probable number (MPN) index. This is an index of the number of coliform bacteria that, more probably than any other number, would give the results shown by the test. It is not a count of the actual number of indicator bacteria present in the sample. MPN test is performed in 3 steps 1.Presumptive test 2.Confirmatory test 3.Completed test Presumptive test The presumptive test is a screening test to sample water for the presence of coliform organisms Principle Separate analyses are usually conducted on five portions of each of three serial dilutions of a water sample. The individual portions are used to inoculate tubes of culture medium that are then incubated at a standard temperature for a standard period of time. The presence of coliforms is indicated by turbidity in the culture medium, by a pH change and/or by the presence of gas. The MPN index is determined by comparing the pattern of positive results (the number of tubes showing growth at each dilution) with statistical tables. The tabulated value is reported as MPN per 100 ml of sample. Technique Water to be tested is diluted serially and inoculated in lactose broth, coliforms if present in water utilizes the lactose present in the medium to produce acid and gas. The presence of acid is indicated by the color change of the medium and the presence of gas is detected as gas bubbles collected in the inverted Durham tube present in the medium. The number of total coliforms is determined by counting the number of tubes giving positive reaction (i.e both color change and gas production) and comparing the pattern of positive results (the number of tubes showing growth at each dilution) with standard statistical tables. Procedure Requirements Medium: Lactose broth or MacConkey broth or Lauryl tryptose (lactose) broth Glasswares: Test tubes of various capacities (20ml, 10ml, 5ml), Durham tube Others: Sterile pipettes Preparation of the Medium Prepare medium (either MacConkey broth or lactose broth) in single and double strength concentration. For untreated or polluted water : Dispense the double strength medium in 5 tubes (10mL in each tube) and single strength medium in 10 tubes (10 mL in each tube)and add a Durham tube in an inverted position. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.  Take 5 tubes of double strength and 10 tubes of single strength for each water sample to be tested. Using a sterile pipette add 10 mL of water to 5 tubes containing 10 mL double strength medium. Similarly, add 1 mL of water to 5 tubes containing 10 mL single strength medium and 0.1 mL water to the remaining 5 tubes containing 10 mL single strength medium. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re- incubate up to 48 hrs. Compare the number of tubes giving a positive reaction to a standard chart and record the number of bacteria present in it. For example, a water sample tested shows a result of 3–2–1 (3 × 10 mL positive, 2 × 1 mL positive, 1 × 0.1 mL positive) gives an MPN value of 17, i.e. the water sample contains an estimated 17 coliforms per 100 ml MPN Water Testing For untreated (polluted) water Take 5 tubes of double strength and 10 tubes of single strength for each water sample to be tested. Using a sterile pipette add 10 mL of water to 5 tubes containing 10 mL double strength medium. Similarly, add 1 mL of water to 5 tubes containing 10 mL single strength medium and 0.1 mL water to the remaining 5 tubes containing 10 mL single strength medium. Incubate all the tubes at 37°C for 24 hrs. If no tubes appear positive re-incubate up to 48 hrs. Compare the number of tubes giving a positive reaction to a standard chart and record the number of bacteria present in it. For example, a water sample tested shows a result of 3–2–1 (3 × 10 mL positive, 2 × 1 mL positive, 1 × 0.1 mL positive) gives an MPN value of 17, i.e. the water sample contains an estimated 17 coliforms per 100 m Confirmatory Test Some microorganisms other than coliforms also produce acid and gas from lactose fermentation. In order to confirm the presence of coliform, a confirmatory test is done. Confirmed Test It is the confirmatory test ensuring the presence of coliform by testing the positive tubes of the presumptive test. The gas produced in the presumptive test does not mean that there must be coliform in the water sample. Many other microorganisms are also present in water, which can give a false presumptive test. Some yeasts and Clostridium species are present in the water, which can ferment lactose by producing both acid and gas. Therefore, it becomes necessary to confirm the presence of coliform in water. A confirmed test can be performed usually in two ways: In brilliant green lactose bile broth (BGLB) In eosin methylene blue agar medium (EMB) Confirmatory test- BGLB test From each of the fermentation tubes with positive results transfer one loopful of medium to: 3 mL lactose-broth or brilliant green lactose fermentation tube- The brilliant green dye in the BGLB medium inhibits the growth of gram- positive bacteria., Incubate the inoculated lactose-broth fermentation tubes at 37°C and inspect gas formation after 24 ± 2 hours. If no gas production is seen, further incubate up to a maximum of 48 ±3 hours to check gas production. Brilliant green lactose broth The formation of gas in lactose broth and the demonstration of Gram-negative, non-spore- forming bacilli in the corresponding agar indicates the presence of a member of the coliform group in the sample examined. The absence of gas formation in lactose broth or the failure to demonstrate Gram-negative, non- spore-forming bacilli in the corresponding agar slant constitutes a negative test (absence of coliforms in the tested sample). EMB test For this inoculum from each positive tube of the confirmatory test is streaked on a plate of EMB or Endo agar. In this process, a loopful of a sample from each positive BGLB tube is streaked onto selective medium like Eosin Methylene Blue agar or Endo’s medium. One plate each is incubated at 37°C and another at 44.5± 0.2°C for 24 hours. Following incubation, all plates are examined for the presence of typical colonies. Coliforms produce colonies with a greenish metallic sheen which differentiates it from non-coliform colonies (show no sheen). The presence of typical colonies on high temperature (44.5 ±0.2) indicates the presence of thermotolerant E.coli. EMB agar test Following incubation, all plates are examined for the presence of typical colonies. Coliforms produce colonies with a greenish metallic sheen which differentiates it from non- coliform colonies (show no sheen). The presence of typical colonies on high temperature (44.5 ±0.2) indicates the presence of thermotolerant E.coli. Uses of EMB agar Isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli. 1.EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples. 2. (presence of E.coli in the river/water sample indicates the possibility of fecal contamination of water so does the presence of other pathogenic enterics). 3.EMB media assists in the visual distinction of Escherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera. 4.Escherichia coli colonies grow with a metallic sheen with a dark center. 5.Aerobacter aerogenes colonies have a brown center, and non-lactose-fermenting gram- negative bacteria appear pink. 6.EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group. 7.For culture of Salmonella and Shigella, selective medium such as MacConkey agar and EMB agar is commonly used. Completed Test It is the final test to ascertain the presence of coliforms. A completed test involves the following steps: Take a loopful of culture from positive confirmed tube. Inoculate a culture either in BGLB medium or by streaking the culture onto the agar slants. Incubate the test tubes for 24-48 hours at 37 degrees Celsius. Observation: Observe the test tubes for gas production in BGLB medium or the growth of a bacterial colony in the agar slants. Result interpretation: The production of gas in the Durham tube of the BGLB fermentation tube confirms the presence of coliform bacteria. The agar slants should be incubated at 37°C for 24± 2 hours and Gram-stained preparations made from the slants should be examined microscopically. The growth of gram-negative, non-sporing rods in the agar slants confirms the presence of coliforms. Uses of MPN test 1. used in estimating microbial populations in soils, waters, agricultural products. 2. useful with samples that contain particulate material that interferes with plate count enumeration methods. 3. is an alternate method to trend environmental monitoring studies. 4. is useful for counting bacteria that reluctantly form colonies on agar plates or membrane filters but grow readily in liquid media. Advantages of Most Probable Number (MPN) Ease of interpretation, either by observation or gas emission Sample toxins are diluted Effective method of analyzing highly turbid samples such as sediments, sludge, mud, etc. Permits samples that cannot be analyzed by membrane filtration. Limitations of Most Probable Number (MPN) Poor accuracy and precision associated with MPN count usually mean that the method is one of last resort — to be considered only when other counting methods are inappropriate. Laborious and expensive in terms of materials, glassware, and incubator space. It has relatively a large margin of error.