Tribhuvan University Institute of Science and Technology ... · Microbiology without whom the experiments and practical assignments wouldn’t be completed as expected. Thanking you

© Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu; Email: [email protected]

Tribhuvan University Institute of Science and Technology

Central Department of Microbiology Kirtipur, Kathmandu

PRACTICAL RECORD FILE (M.Sc. Public Health Microbiology-Second Semester)

SUBMITTED BY:

Name of Student : ………………………………………………………………………

Symbol No. : ………………………………………………………………………

Semester : ………………………………………………………………………

Subject : ………………………………………………………………………

Academic Year : ………………………………………………………………………

2018



Central Department of Microbiology Kirtipur, Kathmandu

M.Sc. Microbiology Public Health Microbiology

Second Semester (Applied Environmental Microbiology)

LAB INSTRUCTORS

Dr. Dev raj Joshi, Lecturer

Mr. Upendra Thapa Shrestha, Lecturer

2018


LIST OF EXPERIMENTS

S. no.

Name of Experiment Page no.

Date of Experiment

Signature (Lab Supervisor)


S. no.

Name of Experiment Page no.

Date of Experiment

Signature (Lab Supervisor)


ACKNOWLEDGEMENTS

Working since many years, I ever felt the necessity of a practical record manual instead of writing on Exercise book for M. Sc. level students. The major drawback seemed in preparing practical file is copying each other from the top to bottom. Since the introduction, principle and methods of an experiment will be similar for all, the main differences will be on processing for different samples, their observation and how they will discuss their own results. With this concept, the practical record manual is designed with inclusive of all basic contents. The students will study the introduction, principle and methods from this practical manual and will perform their experiments. Once they complete their experiments, they will write observation and discuss their own results in this manual. This practice will save time for students on writing each and every experiment and gained practical skills. While preparing this manual, many people directly and indirectly help to complete this manual. I would like to thank all of them. Firstly, I would like to thank my dear students for individual topic preparation; Apsara Khadka (Chloride), Chandri Lama (Ammonia), Kushal Sitaula (Acidity/Alkalinity), Mandira Niraula (Physical parameters; conductivity, total dissolved solid), Muna Khanal (Arsenic), Pratikshya Shrestha (Total Hardness), Rohit Chaudhary (Residual chlorine), Rubina Gurung (Nitrate and nitrite), Sanam Chaudhary (Membrane filter technique-MF), Sarala Nhemaphuki (Most probable number-MPN), Sayara Bista (DO/BOD), Sunita Poudel (COD), Sushila Belbase (Physical parameters; pH, temperature, turbidity) and Sushmita Kuikel (Iron). I highly appreciate Ms. Sayara Bista for her regular updates, helping on designing format and other necessary arrangements. I would also like to thank Dr. Megha Raj Banjara, Head of Department for approving the new system on practical record file and continuous support on this work. My special thanks go to Prof. Dr. Anjana Singh, Associate Prof. Dr. Prakash Ghimire, Ms. Reshma Tuladhar, Dr. Devaraj Joshi and Mr. Nabaraj Adhikari for giving ideas to develop this record file. Finally, I would like to thank all the faculties and staff members of Central Department of Microbiology without whom the experiments and practical assignments wouldn’t be completed as expected. Thanking you.

Compiled and prepared by

Upendra Thapa Shrestha Lecturer

Central Department of Microbiology Tribhuvan University

Email: [email protected] Domain: www.upendrats.blogspot.com

mailto:[email protected]


STANDARD PRACTICES FOR BIOSAFETY 1. The laboratory supervisor must enforce the institutional policies that control access

to the laboratory. 2. Persons must wash their hands after working with potentially hazardous materials

and before leaving the laboratory. 3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing

food for human consumption must not be permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.

4. Mouth pipetting is prohibited; mechanical pipetting devices must be used. 5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken

glassware must be developed and implemented. Whenever practical, laboratory supervisors should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include: a. Careful management of needles and other sharps are of primary importance.

Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.

b. Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.

c. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

d. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps. Plastic ware should be substituted for glassware whenever possible

6. Perform all procedures to minimize the creation of splashes and/or aerosols. 7. Decontaminate work surfaces after completion of work and after any spill or

splash of potentially infectious material with appropriate disinfectant 8. Decontaminate all cultures, stocks, and other potentially infectious materials

before disposal using an effective method. Depending on where the decontamination will be performed, the following methods should be used prior to transport. a. Materials to be decontaminated outside of the immediate laboratory must

be placed in a durable, leak proof container and secured for transport. b. Materials to be removed from the facility for decontamination must be

packed in accordance with applicable local, state, and federal regulations. 9. A sign incorporating the universal biohazard symbol must be posted at the entrance

to the laboratory when infectious agents are present. The sign may include the name of the agent(s) in use, and the name and phone number of the laboratory supervisor or other responsible personnel. Agent information should be posted in accordance with the institutional policy.


Central Department of Microbiology


1

EXPERIMENT NO: 1 Date:

PHYSICAL QUALITY ANALYSIS OF WATER DETERMINATION OF TEMPERATURE, pH, TURBIDITY, CONDUCTIVITY AND TOTAL DISSOLVED SOLIDS IN WATER SAMPLES A. TEMPERATURE

OBJECTIVES:

1. To determine the temperature of water sample INTRODUCTION:

The parameter of temperature is basically important for its effects on the chemistry and biological reaction in the organisms in water. A rise in temperature of the water leads to the speeding up of the chemical reaction in water, reduces the solubility of gases and amplifies the tastes and odors. Water in the temperature range of 7 ℃ to 11℃ has a pleasant taste and is refreshing. At higher temperature with less dissolved gases, the water becomes tasteless and even does not quench the thirst. At elevated temperatures metabolic activity of the organisms increases, requiring more oxygen but at the same time the solubility of oxygen decreases, thus accentuating the stress.

PRINCIPLE: To correct the readings for the differences between the temperature at the time of reversal and time of reading, a small mercury thermometer is also attached in the side of reversing thermometer. This thermometer is called as an auxiliary thermometer. For protection and to eliminate the effect of hydrostatic pressure, the whole unit is kept in thick glass tube, partially evacuated and filled with mercury reservoir of the reversing thermometer to provide better thermal conductivity. The correction factor can be obtained from the following equation.

T = T1 + T, where

T = [{(T1- t) (T1-V0)}/K-100] +I Where; T = corrected temperature of reversing thermometer

T1 = uncorrected reading of reversing thermometer t= temperature from auxiliary thermometer at which reading is taken. V0 =volume of small bulb and of capillary upto 0℃ graduation. K = constant, depending upon the relative expansion of mercury and the type of glass used in thermometer. I = the individual calibration correction for reversing thermometer.

Note: Instead of indulging in tedious calculations every time, ΔT can be obtained from a graph drawn for values of T1 and t in relation to ΔT. REQUIREMENTS:

1. Thermometer 2. Water sample




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METHODS:

1. At the control site the thermometer was placed about 0.5 inches from the bottom or a few inches below the water surface. The thermometer was kept in the water until a constant reading was attained (approximately two minutes).

2. The measurement was recorded in Celsius. 3. The test was repeated and subtracted the upstream temperature from the reference

site from the temperature downstream and recorded the result as temperature changed.

4. The temperature data was collected under similar conditions and using the same

thermometer. Thermometer should be readable to 0.1C. 5. The result was then calculated and interpreted.

OBSERVATIONS: Table 1:

S. No. Sample type Temperature (C)




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B. pH (POTENTIAL HYDROGEN ION) OBJECTIVES:

1. To determine the pH of water sample INTRODUCTION:

pH is the measure of the intensity of acidity or alkalinity and measures the concentration of hydrogen ions in water. It does not measure total acidity or alkalinity. In fact, the normal acidity or alkalinity depends upon excess of H+ or OH-ions over the other, and measured in normality or gram equivalent s of acid or alkali. If free H+ are more than OH- ions, the water shall be acidic, or alkaline the other way around. pH is generally measured on a log scale and equals to negative log of hydrogen ion concentration.

pH = -log10[H+]

= log10 𝟏

[𝐇+]

As the ionic product of water is 1× 10 -14 at 25℃, therefore, a neutral solution will have 1 x 10-7 ions of H+ and OH- each. pH scale ranges from0 to 14 with 7 as neutral, below 7 being acid and above 7 is alkaline REQUIREMENTS:

1. pH meter 2. Water sample

METHODS:

1. First the pH meter was set with a buffer whose value is near to the expected pH of the sample.

2. Buffers of different pH values was made in the laboratory in the following manner: 3. A: Potassium hydrogen phthalate buffer (0.2 g of Potassium hydrogen phthalate was

dissolve in water to prepare 1000 ml of buffer.) 4. B: Phosphate buffer (3.40 g of KH2PO4 and 4.45 g of Na2HPO42H20 was dissolved in

water to prepare 1000 ml of buffer.) 5. C. Borax buffer (3.81 g of Na2B407 10H20 was dissolved in water to prepare 1000 ml of

buffer.) 6. The values of the above buffers at various temperatures are as follows:

Temperature ℃ Phthalate Phosphate

0 4.01 6.98 5 4.00 6.95 10 4.00 6.92 15 4.00 6.90 20 4.00 6.88 25 4.01 6.86 30 4.01 6.8




4


S. No. Sample type pH (At C)




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C. TURBIDITY

OBJECTIVES: 1. To determine the turbidity of water sample

INTRODUCTION:

Turbidity in water is caused by the substances not present in the form of true solution. True solutions have a particle size of less than 10-9 m. Any substance having more than this size will produce a turbidity. Turbidity of water is actually the expression of optical property (Tyndall effect) in which the light is scattered by the particles present in the water. Turbidity in natural water is caused by clay, silt, organic matter, phytoplankton and other microscopic organisms. Turbidity determinations do not correlate with the actual amount of suspended matter as the scattering of light is highly dependent upon the size, shape and refractive index of the particles. Turbidity makes the water unfit for domestic purpose s, food and beverage industries and many other industrial uses. Determination of turbidity is an important objective in removal of the turbidity by coagulation, filtration, etc. in drinking water treatment plants. A reduction in turbidity is associated with a reduction in suspended matter and microbial growth. Turbidity in natural waters resides light penetration of photosynthesis. REQUIREMENTS:

1. Nephelometer (It measures the scattered light at the right angle of the path of incident

light.)

2. Sample tubes (These should be clean and made up of a colorless glass. The tubes

should be free of any scratch or etching.)

3. Stock turbidity suspensions

(Dissolve 1 g of hydrazine sulphate (NH2)2.H2SO4 in distilled water to prepare 100 ml of solution. Dissolve 10 g hexamethylene tetramine (CH2)6N4 in distilled water to prepare 100 ml of solution. Mix 5 ml of each of the above solutions in a100 ml volumetric flask and allow to

stand for 24 hrs. at 25C. Dilute it to 100 ml mark. This is 400 NTU (Nephelometric turbidity units) suspension. This solution can be kept for a month.)

4. Standard turbidity suspension

(Prepare 40 NTU solution by diluting 10 ml of stock solution to 100 ml. This solution should be prepared every week.) METHODS:

1. For handling the Nephelometer, the instructions supplied by the manufactures was

followed.

2. The instrument was set at 100 with 40 NTU standard suspension. In this case, every

division on the scale will be equal to 0.4 NTU turbidity.




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3. The sample was shaken thoroughly and kept it for some time to eliminate the air

bubbles.

4. The sample was put in the Nephelometer sample tube and found out the value on the

scale.

5. If sample was suspected to have more turbidity, it was diluted so that turbidity values

come below 40 NTU and reading was taken.

CALCULATION:

Turbidity, NTU = Nephelometer reading × 0.4 × dilution factor OBSERVATIONS: Table 3:

S. No. Sample type Dilution Factor Nephelometer reading

Turbidity NTU




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D. CONDUCTIVITY

OBJECTIVES: 1. To determine the conductivity of water sample

INTRODUCTION: Conductivity is the measure of capacity of a substance or solution to conduct electric current. As most of the salt present in the water are present in the ionic form, capable of conducting current. Therefore, conductivity is the good and rapid measure of the total dissolved solids. The present of salts and contaminants with waste waters increase the conductivity of the water. Consequently, a sudden rise in conductivity in the water will indicate addition of some pollutants to it. The conductivity is generally reported in mho or mho. The conductivity of distilled water ranges between 1 to 5 mhos. The recent unit of conductivity has been named as Siemens (S) instead of mho. Conductivity, however, is an important criterion in determining the suitability of water and waste water for irrigation. Water having conductivity more than 20 mho have not been found suitable for irrigation. PRINCIPLE: Electrical conductance is the ability of a substance to conduct the electric current. In water, it is the property caused by the presence of various ionic species. It is generally measured with the help of a conductivity meter having a conductance cell containing electrodes of platinum. These electrodes are mounted rigidly and parallel at a fixed distance. Conductance, when measured between the electrodes having the surface area 1 cm and placed at a distance of 1 cm, is called electrical conductivity and is the property of water sample rather than the measuring system. Conductivity is the highly dependent upon temperature and therefore is reported normally at 25° C. REQUIREMENTS:

1. Conductivity meter

2. Thermometer

3. Glassware

METHODS:

1. Prepare a 1:5 soil suspension by taking 20 g of soil in 100 ml or aerated distilled water.

2. Shake a soil suspension mechanically for one hour.

3. Measure the conductivity of the soil suspension with conductivity meter by directly dipping the cell into the suspension.

4. Take temperature of the soil suspension and convert the result at 25°C.

OBSERVATIONS: CALCULATION: Conductivity = observed conductance × cell constant × temperature factor at 25°C




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E. TOTAL DISSOLVED SOLID (TDS)

OBJECTIVES: 1. To determine the TDS of water sample

INTRODUCTION:

1. Total dissolved solids (TDS)

Dissolved solids refer to any minerals, salts, metals, cations, and anions in water. Total dissolved solids (TDS) comprises inorganic salts principally calcium, magnesium, potassium, sodium, bicarbonates, chlorides, and sulfates, and some small amounts of organic matter that are dissolved in water. Dissolved solids do not contain any gas and colloids. Concentration of dissolved solids is an important parameter in drinking water and other water quality standards. They give a particular taste to the water at higher concentration and also reduce its palatability. Total dissolved solids in drinking water originate from natural sources, sewage, urban runoff, industrial waste water and chemical used in the water treatment process and the nature of the piping or hardware used to convey the water i.e., plumbing. In general, the total dissolved solids concentration is the sum of the cations and anions ions in water. Therefore, the total dissolved ions don’t tell us the nature or ion relationship. PRINCIPLE: Total dissolved solids(TDS) are determined as the residue left after evaporation of the filtered sample. It is a measure of the combined content of all inorganic and organic substance contained in a liquid in molecular, ionized or micro granular suspended form. Generally, the operational definition is that the solids must be small enough to survive filtration through a filter with two micrometer pores. Total dissolved solids are normally discussed only for fresh water systems. The principle application of TDS is in the study of water quality for streams, rivers, lakes, although TDS is not generally considered a primary pollutant, it is used as an indication of aesthetic characteristics of drinking water and as an aggregate indicator of the presence of a broad array of chemical contaminants. REQUIREMENTS:

1. Evaporating dish

2. Filter paper

3. Water bath

4. Oven

METHODS:

1. Take an evaporating dish and ignite it at 550±50° C in a muffle furnace for about an

hour, in a desiccators and weigh.

2. Filter the sample through glass fiber filter paper applying the suction.




9

3. Evaporating 100 ml of this filtered sample or more in case the solids are less than 25

mg/L in the pre-weighed evaporating dish on a water bath or a bot plate having

temperature not more than 98° C.

4. Heat the residue at 103-105° C in an oven for one hour and take the final weight after

cooling in desiccators.

OBSERVATIONS: CALCULATION:

𝑻𝑫𝑺 (𝒎𝒈/𝑳) =(𝑨 − 𝑩) × 𝟏𝟎𝟎𝟎 × 𝟏𝟎𝟎𝟎

𝑽

Where; A = Final weight of the dish in g B = Initial weight of the dish in g V = Volume of sample taken in ml Table 4:

S. No. Sample type A (Final Weight) B (Initial weight) Vol. of sample taken (V)

RESULTS AND DISCUSSION:




10

CONCLUSION:

PRECAUTIONS:

REFERENCES:




11


CHEMICAL QUALITY ANALYSIS OF WATER DETERMINATION OF ACIDITY AND ALKALINITY OF WATER OBJECTIVES:

1. To determine the alkalinity of water samples 2. To determine the acidity of water samples

INTRODUCTION: ALKALINITY: Alkalinity of water is its capacity to neutralize a strong acid and is characterized by the presence of all hydroxyl ions capable of combining with the hydrogen ion. Alkalinity in natural water is due to free hydroxyl ions and hydrolysis of salts formed by weak acid and strong base.

𝑨− + 𝑯𝑶𝑯 = 𝑯𝑨 + 𝑶𝑯− When a salt of weak acid and strong base is hydrolyzed it forms the weak acid and strong base. The weak acid is unable to dissociate more, and when the titration is carried out with the strong acid the equilibrium is shifted to the right and all the salt is hydrolyzed. The number of milliequivalents of acid is used in the titration to combine all the hydroxyl ion is called as total equivalents. Most of the alkalinity in natural waters is formed due to of carbon dioxide in water. Carbonates and bicarbonates thus formed, are dissociated to yield hydroxyl ions. Carbonate salts produce double the hydroxyl ions than the bicarbonates. Therefore, total alkalinity caused by a CO2-H20 system can be represented by following equation.

𝑪𝑶𝟐 + 𝑯𝟐𝑶 𝑯𝟐𝑪𝑶𝟑 𝑯𝟐𝑪𝑶𝟑 𝑯+ + 𝑯𝑪𝑶𝟑

−

𝑯𝑪𝑶𝟑− 𝑯+ + 𝑪𝑶𝟑

−𝟐 𝑪𝑶𝟑

−𝟐 + 𝟐𝑯𝑶𝑯 𝑯𝟐𝑪𝑶𝟑 + 𝟐𝑶𝑯− 𝑯𝑪𝑶𝟑

− + 𝑯𝑶𝑯 𝑯𝟐𝑪𝑶𝟑 + 𝑶𝑯−

Total alkalinity =𝑯𝑪𝑶𝟑− + 𝟐𝑪𝑶𝟑

−𝟐 + 𝑶𝑯− + 𝑯+ Alkalinity is also produced by action of water and limestone or chalk. Carbonic acid formed after the dissolution of CO2 is a diprotic acid and it gets dissociated into HCO3

- and CO3-- in two stages. At varying pH, different proportions of these species are

present. Between pH 0.0 and 6.35 almost all the species in the CO2-H20 system are present in form of carbonic acid, between pH 6.35 and 10.33 almost all species in the form of HCO3

-, and between pH 10.33 and 14.0 almost all the species in the form of CO3

--. In the natural and polluted waters, there are many other salts of weak acids such as silicates, phosphates, borates etc. which alkalinity in addition to that of CO3

-- and HCO3-. Naturally

coloured water also contain humates (salts of humic acid and fulvic acid), which also add to the alkalinity of waters.




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Alkalinity in itself is not harmful to human beings, still the water supplies with less than 100mg/L are desirable for domestic use. The alkalinity value is also important in calculating the dose and biocides in water. Alkalinity producing substances such as sodium bicarbonate, are added to check corrosion in soft water supplies. Alkalinity measurements are also important in controlling water and waste water treatment processes. The ratio of alkalinity to that of alkaline earth metals is a good parameter determining the suitability of irrigation waters. All the carbonates and bicarbonates are converted to carbonic acid at pH 3.65, therefore in determination of total alkalinity a suitable indicator changing colour at this pH is required, methyl orange is most appropriate indicator changing its color at this pH. ACIDITY: Acidity of water is its capacity to neutralize a strong base and is mostly due to presence of strong mineral acids, weak acids (carbonic acid, acetic acid) and the salts of strong acids and weak bases (ferrous sulphate, aluminium sulphate etc.). These salts on hydrolysis produce a strong acid and metal hydroxides which are sparingly soluble thus producing the acidity. Addition of waste waters having acidity producing substances also increases the acidity of waters.

𝑭𝒆𝑺𝑶𝟒 + 𝟐𝑯𝟐𝑶 𝑭𝒆 (𝑶𝑯)𝟐 + 𝑯𝟐𝑺𝑶𝟒 𝑨𝒍𝟐(𝑺𝑶𝟒)𝟑 + 𝟔𝑯𝟐𝑶 𝟐𝑨𝒍(𝑶𝑯)𝟑 + 𝟑𝑯𝟐𝑺𝑶𝟒

However, in natural waters most of the acidity is present due to the dissolution of CO2 which forms carbonic acid.

𝑪𝑶𝟐 + 𝑯𝟐𝑶 𝑯𝟐𝑪𝑶𝟑 Determination of acidity is significant as it causes corrosion and influences the chemical and biochemical reaction. PRINCIPLE: A. TOTAL ALKALANITY (CARBONATES AND BICORBONATES) Total alkalinity is the measure of the capacity of water to neutralize a strong acid. The alkalinity in water is generally imparted by the salts of carbonates and bicarbonates, phosphates, nitrates, borates, silicates etc. together with hydroxyl ions in free state. Total alkalinity, carbonates and bicarbonates can be estimated by titrating the sample with a strong acid (HCl or H2SO4), first to pH 8.3 using phenolphthalein as an indicator and then further to pH between 4.2 and 5.4 with methyl orange. In first case, the value is called as phenolphthalein alkalinity (PA) and in second case, it is total alkalinity (TA). Values of carbonates, bicarbonates and hydroxyl ions can be compared from these two types of alkalinities. REQUIREMENTS:

1. HCL (0.1N)

2. Methyl orange indicator ,0.05%

3. Phenolphthalein indicator METHODS:

1. 100 ml of sample was taken in a conical flask and 2 drops of phenolphthalein was added to it.




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2. If the solution remains colorless, PA=0 3. If the color changes to pink after addition of phenolphthalein, titrate it with 0.1 N HCl

until the color disappear at end points. This was phenolphthalein alkalinity. 4. Now 2-3 drops of methyl orange were added to the same sample and titration was

continued further until the yellow color change to pink at end point. This was total alkalinity.

CALCULATION:

PA as CaCO3, mg/L =(𝑨 × 𝑵𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚) 𝒐𝒇 𝑯𝑪𝒍 ×𝟏𝟎𝟎𝟎 ×𝟓𝟎

𝒎𝒍 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆

TA as CaCO3, mg/L ==(𝑩 × 𝑵𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚) 𝒐𝒇 𝑯𝑪𝒍 ×𝟏𝟎𝟎𝟎 ×𝟓𝟎


Where, A = ml of HCl used with only phenolphthalein B = ml of total HCl used with phenolphthalein and methyl orange PA = phenolphthalein alkalinity and

TA = total alkalinity Values of hydroxyl ions, carbonates and bicarbonates can be determined from the values of phenolphthalein and total alkalinity. Table 1:

Result of Titration OH- alkalinity as CaCO3

CO3—alkalinity as

CaCO3 HCO3

- alkalinity as CaCO3

P = 0 0 0 T

P < 1/2T 0 2P T-2P

P = 1/2T 0 2P 0

P > 1/2T 2P-T 2(T-P) 0

P = T T 0 0

Note: P = Phenolphthalein Alkalinity, T = Total Alkalinity OBSERVATIONS: Table 2:

S. No.

Vol of sample taken

A (ml of HCl used with only

phenolphthalein)

B (ml of total HCl used with phenolphthalein and methyl orange)

PA TA




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B. ACIDITY Titration method PRINCIPLE: Acidity of the water is its capacity to neutralize a strong base to a fixed Ph. It is caused by the presence of strong mineral acids, weak acids, and hydrolyzing salts of strong acids. However, in natural unpolluted freshwaters, the acidity is mostly due to the presence of free CO2 in the form of carbonic acid. Acidity can be determined by titrating the sample with a strong base such as NaOH using methyl orange or phenolphthalein as an indicator. If the sample has strong mineral acids and their salts, it is titrated first to pH 3.7 using methyl orange as an indicator. This is called as methyl orange acidity. If the sample is titrated directly to pH 8.3 using phenolphthalein, the resultant value is total acidity. The results of acidity should always be accompanied by the end point pH The titration method is suitable mainly for the samples which are colorless. The original color of the sample may interfere with indicator colours. REQUIREMENTS:

1. NaOH (0.05 N) 2. Methyl orange indicator 3. Phenolphthalein indicator

METHODS:

1. 100 ml of colorless sample was taken in a conical flask and 2-3 drops of methyl orange was added.

2. If the solution turns yellow, it indicates absence of methyl orange acidity. In case the content turns pink, it was titrated with 0.05N NaOH. At the end point color changed from pink to yellow.

3. Now 2-3 drops of phenolphthalein was added to the same sample and titrated further with NaOH until the contents turn pink

CALCULATION:

Methyl orange acidity, =𝑨 ×𝑵 𝑶𝑭 𝑵𝒂𝑶𝑯 ×𝟏𝟎𝟎𝟎 ×𝟓𝟎


mg/L as CaCO3

Phenolphthalein acidity,=𝑩 × 𝑵 𝒐𝒇 𝑵𝒂𝑶𝑯 ×𝟏𝟎𝟎𝟎×𝟓𝟎


mg/L as CaCO3

Total acidity to pH 8.3,=(𝑨+𝑩)×𝑵 𝒐𝒇 𝑵𝒂𝑶𝑯 ×𝟏𝟎𝟎𝟎 ×𝟓𝟎


mg/L as CaCO3 Where, A = Volume of NaOH used with methyl orange in titrating the sample to pH 3.7

B = Volume of NaOH used with phenolphthalein in titrating the sample from pH 3.7 to pH 8.3.




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S. No.

Vol of Sample

A (Vol of NaOH used with methyl orange)

B (Vol of NaOH used with phenolphthalein)

Methyl orange acidity

Phenolphthalein acidity

Total acidity





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CONCLUSION:

PRECAUTIONS:

REFERENCES:




17

EXPERIMENT NO: 3 Date: DETERMINATION OF DISSOLVED OXYGEN (DO) IN WATER OBJECTIVES:

1. To determine concentration of dissolved oxygen in water INTRODUCTION:

Dissolved oxygen is a paramount important to all living organisms and is considered to be

lone factor, which to a great extent can reveal nature of whole aquatic system at a glance.

Dissolved oxygen measures the amount of gaseous oxygen dissolved in aquatic life. DO level

depends on the physical, chemical and biological activities of water body. Presence of Do in

water may be due to following phenomena:

a. Direct diffusion of air

b. By photosynthetic activities of aquatic autotrophs

c. By splashing, tumbling of water, O2 enters to the water.

Direct diffusion of air is purely physical process and depends on the solubility under influence

of temperature, salinity, water movements etc. Whereas photosynthetic activities are

biological process and depends on availability of light and rate of metabolic process.

Dissolved oxygen is utilized by microorganisms, aquatic animals like fish etc. to some extent.

If the concentration of Do falls below 5mg/l then, aquatic life is under stress. Minimum

amount of dissolved oxygen concentration that can support large diversity of fish population

is 3-5 mg/l.

PRINCIPLE:

Azide iodometric method is modification of Winkler's iodometric method, in this method,

different types of chemicals such as fixative I and fixative II are used. Fixative fixes the

Dissolved oxygen (DO). Fixative I is manganese sulphate, MnSO4 and fixative II is iodine azide

solution. Besides these chemicals, this test uses conc. H2SO4, sodium thiosulphate (Na2S2O3)

and starch. MnSO4 reacts with alkali to form white ppt of Mn(OH)2. This Mn(OH)2 in presence

of oxygen gets oxidized to brown coloured manganic oxide. On addition to conc.H2SO4, basic

manganic ion (Mn++) are reduced by iodine ions (I-), which gets converted into iodine

equivalent to that of DO present in water sample. Finally, liberated iodine is titrated with

standard Na2S2O3 solution using starch as indicators.

Reaction involved:

MnSO4+ NaOH Mn(OH)2 + Na2SO4

(Manganese hydroxide)

Mn(OH)2 + O2 MnO(OH)2 (Brown ppt)

MnO(OH)2 + 2H2SO4 + 2KI MnSO4 + 3H2O + I2

Na2S2O3 + I2 Na2S4O6 + 2 NaI




18

CALCULATION:

The dissolved oxygen (DO) of the water can be calculated as:

𝑫𝑶 (𝒎𝒈/𝑳) =(𝒎𝒍 × 𝑵)𝒐𝒇 𝒕𝒊𝒕𝒓𝒂𝒏𝒕 × 𝟖 × 𝟏𝟎𝟎𝟎

𝑽𝟐(𝑽𝟏 − 𝑽

𝑽𝟏 )

Where, ml = Volume of thiosulphate consumed

N = Normality of thiosulphate solution (0.025N)

V1 = Volume of sample in bottle after placing stopper (300ml)

V2 = Volume of part of sample titrated (100ml)

V = Volume of MnSO4 and alkaline I2 azide solution used (4ml)

REQUIREMENTS:

1. BOD bottle 2. Burette 3. Pipettes 4. Conical flask 5. Measuring cylinder 6. Water sample 7. Conc. H2SO4 8. 0.25 N sodium thiosulphate 9. Alkaline I2 azide solution 10. Starch solution (1%) 11. Manganous sulphate solution

REAGENTS PREPARATION:

1. Sodium thiosulphate, 0.025N: (Dissolve 24.82g of Na2S2O3 H2O in boiled distilled

water and make up the volume to 1 liter. Add 0.4g of borax or a pallet of NaOH as

stabilizer. This is 0.1N stock solution. Dilute it to 4 times with boiled distilled water to

prepare 0.025N solution. Keep in a brown glass stoppered bottle).

2. Alkaline potassium iodide solution: (Dissolve 100g of KOH and 50g of KI in 200ml of

boiled distilled water).

3. Manganous sulphate solution: (Dissolve 100g of MnSO4.4H2O in 200 ml of boiled

distilled water and filter).

4. Starch solution: (Dissolve 1g of starch in 100ml of warm water (800C-900C) distilled

water and add a few drops of formaldehyde solution).

5. Alkali iodide azide solution: [(a). Dissolve 500g of NaOH or 700g of KOH and 150g of

KI in distilled water to make 1 litre solution (b) Dissolve 10g of NaN2 in 40 ml of distilled

water. Mix the two solutions (a) and (b)].




19

METHODS:

1. BOD bottle should be cleaned and dried. 2. It should be filled with water sample without bubbling (300ml). 3. 2ml of MnSO4 should be added using pipette dipping it into the bottom of the bottle. 4. 2ml of alkaline I2 azide solution should be added to the bottle using pipette from the

top. Formation of brown ppt should be observed. 5. The stopper of the bottle should be tightened and ppt should be mixed thoroughly. 6. The ppt should be allowed to settle. 7. 2ml of conc. H2SO4 should be added along the side of the bottle. 8. The stopper should be replaced and bottle should be inverted for 2- 3 times. 9. The ppt should dissolve and solution should turn yellow within 1 hour. 10. 100 ml of solution should be taken in the flask and titrate with 0.025N standard

Na2S2O3 with addition of 1ml of starch in the solution and the colour of the solution changes to blue.

11. The end point is indicated when the initial dark blue colour changes to colourless, (Note the burette reading. Repeat the procedure to obtain concurrent reading).

12. Calculate DO by using above formula. OBSERVATIONS: Table 1:

S.no Volume of titrant (MnSO4 + alk. iodide azide solution)

Burette reading Difference Concurrent reading Initial Final





20

CONCLUSION:

PRECAUTIONS:

REFERENCES:




21

EXPERIMENT NO: 4

Date: DETERMINATION OF BIOCHEMICAL OXYGEN DEMAND (BOD) OF WATER OBJECTIVES:

1. To determine biochemical oxygen demand (BOD) of water

INTRODUCTION:

Biochemical oxygen demand (BOD) is amount of oxygen utilized by the microorganisms in

stabilizing the organic matter. On an average basis, demand for oxygen s proportional to the

amount of organic matter present, which are to be degraded aerobically. It is the measure

which approximates the amount of the oxidizable organic matter present in the solution and

BOD value is used as measure of waste strength. It is important to know the amount of organic

matters present in waste and the quantity of oxygen required for its stabilization. BOD values

are useful in process design as well as measurement of treatment plant efficiency and

operation. It is useful in stream pollution control management and in evaluating the self-

purification capacities of the stream.

For complete degradation of organic waste 20-30 days' time is required which is not

practically suitable, so BOD test is designed at 5 days incubation at 200C. During this period,

simple carbonaceous matter that is glucose is completely oxidized up to 60% and that of

sewage is 40%. BOD value cannot be revealed for total organic load as the waste material may

contain non-degradable matter as well. BOD for nitrifying bacteria at 5oC incubation can be

considered as negligible due to very slow nitrification process.

PRINCIPLE:

Biochemical oxygen demand (BOD) is the measure of the degradable organic material present

in a water sample and can be defined as the amount of oxygen required by the

microorganisms in stabilizing the biologically degradable organic matter under aerobic

conditions.

The principle of the method involves measuring the difference of the dissolved oxygen of the

sample after incubating it for 5 days at 200C.

Biochemical oxygen demand (BOD) is calculated as:

BOD, mg/L = (D0 D5) × dilution factor

Where, D0 = Initial D0 in the sample

D5 = DO after 5 days

If the sample contains free residual chlorine it is necessary to remove it due to its toxic effects

on microorganisms of the sample. For removal of residual chlorine take 50 ml of the sample

and acidify with 10ml acetic acid. Add about 1 gm KI. Titrate with Na2S2O3 (0.025N) using




22

starch as an indicator. Calculate the volume of Na2S2O3 required per ml of the sample and

add accordingly to the sample to be tested for BOD.

REQUIREMENTS:

1. Same as in DO Experiment

METHODS:

1. Two BOD bottles are taken and filled with water samples without bubbling. 2. One of the bottle is incubated at 200C for 5 days and DO of another bottle is calculated

(DO0). 3. After 5 days of incubation DO of sample is determined. 4. Calculation of BOD is done.

OBSERVATIONS: Table 1: For D0



Table 2: For D5







23

CONCLUSION:

PRECAUTIONS:

REFERENCES:




24

EXPERIMENT NO: 5

Date: DETERMINATION OF CHLORIDE IN WATER OBJECTIVES:

1. To determine chloride in water

INTRODUCTION:

Chloride occurs naturally in all types of water. In natural fresh waters, however, its concentrations remain quite low and is generally less than that of sulfate and bicarbonates. The most important source of chlorides in the water is the discharge of domestic sewage. Man, and others animal excreta very high quantities of chlorides together with nitrogenous compound. About 8-15gram of NaCl is excreted by a person per day. Therefore, the chloride concentration serves as an indicator of pollution by sewage. Industries are also important sources of chlorides.

Chlorides are highly soluble with most of the naturally occurring cations and does not precipitates, sediment and cannot be removed biologically in treatment of waste s.

It is harmless upto1500mg/L concentration but produces a salty taste at 200-250mg/L level. It can also corrode concrete by extracting calcium in the form of calcide. Magnesium chloride water generates hydrochloric acid after heating, which is also highly corrosive and create problems in boilers.

PRINCIPLE:

Silver nitrate reacts with chloride to form very slightly soluble white precipitate of AgCl. At the end point when all the chlorides get precipitated, free silver ions react with chromate to form silver chromate of reddish brown color.

REQUIREMENTS:

1. Silver nitrate 0.02N: (Dissolve 3.400gm of dried AgNO3 (A.R) in distilled water to make 1 liter of solution and keep in a dark bottle).

2. Potassium chromate, 5%




25

METHODS:

1. Take 50ml of sample in a conical flask and add 2ml of K2CrO4 solution.

2. Titrate the contents against 0.02N AgNo3 until a persistent red tinge appears.

CALCULATION:

𝑪𝒉𝒍𝒐𝒓𝒊𝒅𝒆 (𝒎𝒈/𝑳) =(𝒎𝒍 × 𝑵) 𝒐𝒇 𝑨𝒈𝑵𝑶𝟑 × 𝟏𝟎𝟎𝟎 × 𝟑𝟓. 𝟓

𝑽𝒐𝒍. 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆

OBSERVATIONS

Table 1:

S. No. Vol of sample taken (ml) Vol of K2CrO4 Vol of AgNO3

consumed Chloride in mg/L





26

CONCLUSION:

PRECAUTIONS:

REFERENCES:




27

EXPERIMENT NO: 6 Date: DETERMINATION OF AMMONIA IN WATER OBJECTIVES:

1. To determine the ammonia in water sample INTRODUCTION: The presence of ammonia (NH3) in water is due to organic matter decomposition. The concentration of ammonia is increased by metabolic activity of microorganism, industrial gas and disinfection process using chloramines. When water is contaminated with sewage, the concentration of ammonia is increased in water. If ammonia is rich in water, some period has been lapsed during pollution. If only NO3 is present, pollution has been occurred at remote time. During this period all NH3 is oxidized to NO3 and a very small extent of NH3 remain in water.

Surface Water – 0.2 mg/lit Ground Water – 3 mg/lit

NH3 is toxic to living organisms. Toxicity increases with increases with increase in pH. Toxicity is directly proportional to pH. In low pH, NH3 is converted into NH4

+ which is less toxic than NH3 gas (at high pH). I. COLORIMETRIC PRINCIPLE: Nessler’s reagent (potassium mercuric iodide) reacts with ammonia in water sample to give pale yellow to brown depending upon the amount of ammonia present. The color obtained can be determined colorimetrically. Most of the natural waters and wastewater have interfering substances therefore, the steam distillation of ammonia becomes essential.

2(2K2 +HgI2) + 3KOH + 2NH3 → (NH2)2. HgOH. HgI (brown) + 7KI + 2H20

REQUIREMENTS: 1. Distillation unit: (There are a number of designs available. The most common

distillation assembly is Micro-Kjeldahl distillation unit). 2. Standard ammonia solution: (Dissolve 3.819g of anhydrous NH4Cl in distilled water to

prepare 1 liter of solution. This solution contains 1000mg/l NH3-N. Dilute this solution 100 times (10 to 1000 ml) to prepare the solution containing 10mg/l NH3-N).

3. Sulfuric acid, 0.04N: (Add 2 ml of (1:1) H2SO4 (sp. Gr. 1.84) to 1 liter of distilled water). 4. Borax buffer: (Add 4g of Na2B4O7. H2O to 100 ml of distilled water. Heat to dissolve the

crystals). 5. Nessler’s reagent (potassium mercuric iodide)(Dissolve 25g of mercuric iodide and

20gm of KI in 500ml of ammonia distilled water. 6. Dissolve 100gm NaOH in 500ml distilled water, store these two solutions in brown

glass air tight stopped bottles. Mix (1+1) just before use.




28

METHODS: 1. Take 50ml of sample in the distillation assembly through tap A and add 1ml of borax

solution. 2. Put 2.5ml of 0.4N H2SO4 in a 100ml conical flask and place below the condenser so

that the tip of the outlet dips into the acid. 3. Keep the boiling flask on the heater to pass the steam into the sample through

chamber. 4. Ammonia will be distilled of and collects in the sulfuric acid as (NH3)2SO4. Continue the

distillation until nearly 40ml of distillate is collected. 5. Remove the flask having distillate. Cool the boiling flask so that all the waste contents

will be sucked into the chamber. 6. Remove the waste contents through tap B. 7. Run the blank with distilled water using same quantity of the chemicals. 8. Make up the volume of distillate to 50ml and add ml of Nessler’s reagent. A brown

color will have developed. 9. Measure the absorbance at 425nm. 10. Prepare the standard curve between 0.05 to 2.0mg/l of NH3-N by diluting the standard

NH3-N solution. CALCULATION: Find out the concentration of NH3-N directly from the standard curve. II. VOLUMETRIC METHOD PRINCIPLE: Ammonia after distillation is dissolved in boric acid and mixed indicator and can be titrated with HCl. Boric acid is so weak an acid that it does not interfere with acidimetric titration. REQUIREMENTS:

1. Hydrochloric acid, 0.01N: (Dilute 0.1 N HCl to 10 times (100- 1000 ml). 2. Boric acid and Mixed Indicator: (Prepare 4% solution of H3BO3 by dissolving 4g boric

acid in 100ml warm distilled water. Mix alcoholic solutions by bromocresol green (0.5%) and Methyl red (0.1%) in 2:1 ratio. Add 5ml if mixed indicator in 100ml of boric acid. If necessary (only when the color becomes blue), adjust the pH with 0.01N HCl until color just turns faint pink to brown.

3. Borax buffer: (Add 4g of borax to 100 ml of distilled water and heat to dissolved the crystals).

METHODS:

1. Set the micro-Kjeldahl assembly and distil of ammonia (see colorimetric determination of NH3) in 5ml of boric acid and mixed indicator instead of H2SO4.

2. Remove the distillate which turns blue due to dissolution of NH3, and titrate with 0.01N HCl. At the end point, contents turn brown to faint pink.

3. Run a blank with same amount of the chemicals.




29

CALCULATION:

𝑁𝐻3 − 𝑁 (𝑚𝑔/𝐿) =(𝐴 − 𝐵) × 𝑁 𝑜𝑓 𝐻𝐶𝑙 × 1000 × 14

𝑉𝑜𝑙. 𝑜𝑓

Where, A= ml of HCl used with sample, B = ml of HCl used with blank OBSERVATIONS: Table 1:

S. No. Sample Vol. of sample (ml)

A (ml of HCl used with sample)

B (ml of HCl used with blank)

Concentration of NH3 (mg/l)





30

CONCLUSION:

PRECAUTIONS:

REFERENCES:




31

EXPERIMENT NO: 7 Date: DETERMINATION OF NITRATE AND NITRITE CONCENTRATION IN WATER OBJECTIVES:

1. To determine the nitrate concentration in water sample 2. To determine the nitrite concentration in water sample

INTRODUCTION:

Nitrate and nitrite are naturally occurring ions that are part of nitrogen cycle. The nitrate

(NO3) is the stable form of combined nitrogen for oxygenated systems; although chemically

unreactive it can be reduced by microbial action. The nitrite ion (NO2) contains nitrogen in a

relatively unstable oxidation state; chemical and biological processes can further reduce

nitrite to various compound or oxidize it to nitrate. The most important source is the

biological oxidation of organic nitrogenous substances. Nitrogen is the nutrient applied in the

largest quantities for lawn and garden care and crop production. Nitrogen occurs naturally in

the soil in organic forms from decaying plant and animal residues. Bacteria in the soil convert

various forms of nitrogen to nitrate, a nitrogen/oxygen ion (NO3) and nitrite (NO2

) is the

intermediate form. This is desirable as the majority of the nitrogen used by plants is absorbed

in the nitrate form.

Nitrate in groundwater may result from point sources such as sewage disposal systems and

livestock facilities, from non-point sources such as fertilized cropland, parks, golf courses,

lawns, and gardens, or from naturally occurring sources of nitrogen. Large amounts of nitrate

in drinking water are a cause of disease called methemoglobinemia, a blood disorder primarily

affecting infants under six months of age. Also, because nitrate contamination can be related

to human, animal, or industrial waste practices, excessive levels of nitrate in drinking water

may indicate potential for the presence of other types of contaminants, which may cause

health problems. Methemoglobinemia is a condition in which the ability of the red blood cells

to carry oxygen is reduced. The acutely poisoned person will have a blue discoloration of the

skin due to the reduction of oxygen in the blood system and must be attended by a physician

immediately.

These are usually monitored in water supplies, as they are potentially hazardous to health.

Maximum admissible concentration for nitrate is 50mg/L. This value is adequate to protect

bottle-fed infants against methemoglobinemia. Maximum admissible concentration for

nitrite is 3mg/L for short-term exposure but for long time exposure the value for nitrite is

0.2mg/L.




32

A. NITRATE

I. BRUCINE METHOD

PRINCIPLE:

Nitrate and brucine react to produce a yellow colour, the intensity of which can be measured

at 410nm. The reaction is highly dependent upon the heat generated during the test.

However, it can be controlled by carrying out the reaction for a fixed time at a constant fixed

temperature. The method is suitable for the sample having a very wide range of salinity.

REQUIREMENTS:

Reagents:

1. Brucine -sulfanilic acid solution: Dissolve 1g brucine sulphate and 0.1g of sufanilic acid

in about 70ml of hot distilled water. After addition of 3ml conc. HCl, make up the

volume to 100ml. The pink colour develops slowly, doesn’t affect the sensitivity.

2. Sulphuric acid solution: Add 500ml H2SO4 in conc.125ml distilled water and cool.

3. Sodium acid solution: Dissolve 5.0g NaAsO8 in distilled water and make 1 liter of

solution.

4. Standard nitrate solution (1mg N/L): Dissolve 0.722g of KNO3 in distilled water and

makeup the volume to 1 liter. This solution contains 100mg N/L. Dilute it to 100 times

to prepare a solution having 1mg N/L(10-1000ml).

METHODS:

1. Free chlorine interferes with the nitrate determination. If the sample is having residual

chlorine, remove it by addition of 0.05ml (one drop) of sodium arsenate solution for

each 0.1mg of chlorine. Add one drop in excess to a 50ml sample portion.

2. Take 10ml of sample or an aliquot diluted to 10ml in a 50ml test tube.

3. Put all the tubes in a wire rack.

4. Place the rack in cool water bath and add 2ml of NaCl solution.

5. Add 10ml of H2SO4 solution after mixing the contents thoroughly swirling by hand.

6. Add 0.5ml brucine reagent and mix thoroughly.

7. Place the rack in a hot water bath with boiling water, exactly for 20 minutes.

8. Cool the contents again in a cold-water bath and take the reading at 410nm.

9. Find out the concentration of NO3-N from the standard curve.

10. Prepare a standard curve between concentration and absorbance by taking the

dilutions from 0.1 to 1.0mg N/L at the interval of 0.1, employing the same procedure

as for the sample.




33

II. PHENOL DISULFONIC ACID METHOD

PRINCIPLE:

Nitrate reacts with phenol disulfonic acid to form a nitro-derivative which in alkaline medium

develops a yellow colour. The concentration of NO3 can be determined colorimetrically, since

the colour so formed obeys the Beer's law.

REQUIREMENTS:

1. Phenol disulfonic acid: Dissolve 25g of white pure phenol in 150ml of conc.H2SO4 and

add 75ml of fuming H2SO4. Heat for two hours on a water bath and keep in a dark

bottle. In place of fuming acid 85ml of conc.H2SO4 can also be added.

2. Silver sulphate solution: Dissolve 4.4g of Ag2SO4 in distilled water to prepare 1litre of

solution.

3. Liquid ammonia, 30%

4. Standard nitrate solution.

METHODS

1. Take 50ml of filtered sample or an aliquot containing not more than 1mg/L of NO3-N

in a conical flask.

2. Add an equivalent amount of Silver sulphate solution to remove chlorides (1 mg/L

Cl=1ml Ag2SO4 solution).

3. Heat slightly and filter the precipitate of AgCl.

4. Evaporate the filtrate in a porcelain basin to dryness.

5. Cool and dissolve the residue in 2ml phenol disulfonic acid and dilute the contents to

50ml.

6. Add 6ml of liquid ammonia to develop a yellow colour.

7. Take the reading at 410nm.

8. Calculate the concentration of nitrate nitrogen from the standard curve.

9. Prepare the standard curve between concentration and absorbance from 0.0mg N/L

to 1.0mg N/L at the interval of 0.1. Find absorbance of the standard solution using the

same procedure described for the sample except the removal of the chlorides as in

steps 2-3.




34

B. NITRITE

PRINCIPLE:

Nitrite forms a diazonium salt with sulphanilic acid in acid medium (2.0-2.5 pH), which

combines with -naphthylamine hydrochloride to form a pinkish dye. The colour so produced

obeys Beer's law and can be determined colorimetrically.

REQUIREMENTS:

1. Disodium ethylene diamine tetra acetic acid (Na2EDTA) solution: Dissolve 500mg of

disodium salt EDTA in distilled water to make 100ml solution.

2. Sulphanilic acid solution: Dissolve 600mg of sulphanilic acid in 70ml of hot distilled

water and add 20ml of concentrated HCl after cooling and dilute to 100ml.

3. -naphthylamine hydrochloride solution: Dissolve 600mg of -naphthylamine

hydrochloride in distilled water to which 1ml concentrated HCl has been added. Dilute

the contents to 100ml and place in a cool place. If a precipitate occurs after few days,

the reagent can be used further by filtering the solution.

4. Sodium acetate solution: Dissolve 16g of anhydrous CH3COONa or 27.2g of

CH3COONa. 3H2O in distilled water to prepare 100ml solution.

5. Standard nitrite solution (1mg/L NH2-N): Dissolve 1.232g NaNO3 in distilled water

and dilute to 1 litre (250 mg/L NO2_N). Dilute this solution 250 times (4-1000ml) to

prepare standard solution having 1mg/L NO2_N.

METHODS:

1. Take 50ml of colourless filtered sample not having more than 1.0mg/L NO2_N in a

conical flask. The colour can be removed by activated charcoal in case of coloured

samples.

2. Add 1ml of each EDTA, sulphanilic acid, -naphthylamine hydrochloride and sodium

acetate solutions in sequence.

3. A wine red colour will appear in the presence of nitrites. Take the reading at 520nm.

4. Compare the absorbance with the standard curve to calculate the nitrite content.

5. Prepare the standard curve between 0.0 to 1.0mg NO2.N/L at the interval of 0.1

employing the same procedure as for the sample.




35

OBSERVATIONS:

Table 1:

S. No. Sample OD520 of standard

solutions

OD520 of sample

solutions

Inference

1.

2.

3.

4.





36

CONCLUSION:

PRECAUTIONS:

REFERENCES:




37

EXPERIMENT NO: 8 Date: DETERMINATION OF TOTAL HARDNESS OF GIVEN WATER SAMPLE OBJECTIVES:

1. To determine the total hardness of water sample

INTRODUCTION: Hardness of water is mainly due to the presence of Calcium and magnesium ions and some extent by other cations such as Sr, Zn, Al, Mn etc. Among anions HCO3

- CO3-, Cl-, and SO4

-- are responsible for hardness of water. According to ions, hardness can be classified as

1) Temporary Hardness: due to HCO3- and CO3

-- of calcium and magnesium.

2) Permanent Hardness: due to Cl- and SO4-- of calcium and magnesium.NO3 is also

responsible.

If the concentration of SO4-- exceeds 500 ppm, it causes bitter taste. SO4

-- along with Cl-

interfaces the normal activity of intestine, depending upon the concentration of hardness.

Water can be classified as

1) Soft water < 50 ppm

2) Moderate hard water 50-150 ppm

3) Hard water 150-300 ppm

4) Very hard water > 300 ppm

Ca2+ is found abundantly in natural water due to leaching from lucks continuously into water. Depending upon the Ca concentration in water, the concentration of in water may be different. It is an important nutrition to organism so it doesn’t have health hazard even if exceeds at 1500 ppm. Calcium can be intoxicating the toxicity of Pb, Zn and KCl. But the increased concentration of calcium in water can increase the hardness of water so not applicable for domestic or industrial purposes. Mg++ is less abundant than Ca in natural water. Important source for Mg are rocks, sewage and industrial waste. If concentration of Mg exceeds 500 ppm, it imports on unpleasant taste and also increases the hardness of water. The hardness of water can be determined by either EDTA method or calculation.




38

A. EDTA METHOD

PRINCIPLE: The total hardness of water is determined by complexometric titration using EDTA as a complexing agent in presence of eriochrome black T indicator.

Ca2+ +H2Y CaY + 2H+

Mg2+ + H2Y MgY +2H+ The Ca-complex formed by reacting Ca2+ and EDTA is more stable than the Mg-complex (MgY). On the other hand, the Mg-In complex with eriochrome black indicator is more stable than Ca-In complex but it is less stable than Mg- EDTA complex (MgY). When the indicator is added in hard water Mg-In complex gives wine red colour. When hard water buffered at the pH 10 is titrated with the standard EDTA solution, first of all Ca and then Mg and finally Mg-In complex react with EDTA to give free indicator imparting blue colour solution.

Mg-In + H2Y MgY + H2In (blue) When the titration is carried out without boiling the given sample of water, the total hardness is determined but the titration after boiling gives the permanent hardness only. The hardness of water is expressed in parts of CaCO3 in millions of parts of water.

1 mol of EDTA ≡ 1 mol of Ca2+ ≡ 1 mol of CaCO3 REQUIREMENTS

1. Glassware: Conical flask, burette 2. EDTA solution, 0.01M: (Dissolve 3.723 g of disodium salt of EDTA in distilled water to

prepare 1 litre of solution. Store in polyethylene or pyrex bottle). 3. Buffer solution: (Dissolve 16.9 g ammonium chloride (NH4Cl) in 143 ml of

concentrated ammonium hydroxide (NH4OH). Dissolve 1.179 g of disodium EDTA and 0.780 g of MgSO4.7H2O in 50 ml distilled water. Mix both above solutions and dilute to 250 ml with distilled water).

4. Eriochrome Black T indicator: (Mix 0.40 g of Eriochrome Black T, with 100 mg NaCl (A.R) and grind).

5. Sodium sulphide solution: (Dissolve 5.0 g of Na2S.9H20 or 3.7 g Na2S.5H2O in 100 ml of distilled water. Tightly close the bottle to prevent oxidation).

6. Other: Water sample, burette stand. METHODS:

1. First of all, 50 ml of sample was taken in conical flask. If the sample is having higher

calcium, a small volume was taken and diluted to 50 ml.

2. 1 ml of buffer solution was taken.

3. If the sample is having higher amount of heavy metals, 1 ml of Na2S solution was

added.

4. 100-200 mg of Eriochrome Black T indicator was added due to which the solution

turned into wine red.

5. The content was titrated against EDTA solution till colour changed from wine red to

blue.




39

6. Process was continued until concurrent reading was obtained.

CALCULATION

Hardness as (mg/L CaCO3) =𝒎𝒍 𝒐𝒇 𝑬𝑫𝑻𝑨 × 𝟏𝟎𝟎𝟎



S. No. Volume of water sample (ml)

Burette reading Concurrent Reading Initial Final Difference

1

2

3

B. CALCULATION METHOD

PRINCIPLE:

This method is used when the sample is having other cations in appreciable amount producing

the hardness. The hardness was found out by multiplying the concentrations of various

cations by their respective factors and the cations were converted to their respective

equivalents in mg/L CaCO3.

Cations Factor Cations Factor

Ca 2.498 Al 5.564

Mg 4.116 Zn 1.531

Sr 1.142 Mn 1.822

Fe 1.792

For this method first of all, the concentration of cation should be calculated. Calcium and magnesium form a complex of wine red colour with Eriochrome Black T at pH 10. The EDTA has got a strong affinity for Ca2+ and Mg2+, the former complex is broken down and a new complex of blue colour is formed. The value of Mg2+ can be obtained by subtracting the value of calcium from the total of Ca2+ + Mg2+.

I. CALCIUM

REQUIREMENTS:

1. EDTA solution, 0.01M

2. Sodium hydroxide,1N




40

3. Dissolve 40 g of NaOH in distilled water and dilute to 1 litre. 4. Murexide indicator: Mix 0.2 g of ammonium purpurate with 100 g and grind.

METHODS:

1. 50 ml sample was taken in a conical flask. If the sample is having higher alkalinity,

smaller volumes was used diluted to 50 ml.

2. 2 ml of NaOH solution was added in the sample.

3. 100-200 mg of murexide indicator was added and a pink color was developed.

4. The mixture was titrated against EDTA solution until the pink colour changed to

purple.

5. For better judgement of end point, the purple color was compared with the distilled

water blank titration end point.

CALCULATION:

Calcium, mg/L = =𝑿 × 𝟒𝟎𝟎.𝟖


Where, X = volume of EDTA used.

II. MAGNESIUM

REQUIREMENTS: 1. EDTA solution, 0.01M 2. Buffer solution 3. Eriochrome Black T indicator

METHODS:

1. The volume of EDTA solution in calcium determination was found out.

2. The volume of EDTA used in hardness determination with same volume of the sample

as taken in the calcium determination was found out.

CALCULATION:

a) Mg2+ (mg/L) =(𝒀− 𝑿) × 𝟒𝟎𝟎.𝟖

𝑽𝒐𝒍. 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 ×𝟏.𝟔𝟒𝟓

Where, Y = EDTA used in hardness determination

X = EDTA used in calcium determination for the same volume of the sample.




41

b) Mg2+ (mg/L) = Total hardness (as mg/L CaCO3) – Calcium hardness (as mg/L CaCO3)

× 0.244

Where, Calcium hardness (as mg/L CaCO3) = Ca, mg/L × 2.497 RESULTS AND DISCUSSION:




42

CONCLUSION:

PRECAUTIONS:

REFERENCES:




43

EXPERIMENT NO: 9

Date: DETERMINATION OF RESIDUAL CHLORINE OF GIVEN WATER SAMPLE OBJECTIVES:

1. To determine the residual chlorine of water sample

INTRODUCTION: When chlorine is added to water some of the chlorine reacts first with organic materials and metals in the water and is not available for disinfection (this is called chlorine demand of water). The remaining concentration after the chlorine demand is accounted for is called total chlorine. Total chlorine is further divided into:

i. The amount of chlorine that has reacted with nitrates and is unavailable for

disinfection which is called combined chlorine and

ii. The free chlorine which is the chlorine available to inactivate disease causing

organisms and thus measure to determine potability of water.

Chlorine can react with naturally occurring compounds in water s to produce compounds known as disinfection by products (DBPs). Ex: Trihalomethane cause Health hazards, slow brain activity, liver, kidney, cancer and heart diseases. Haloacetic acid (HAAS) causes rectal, colon and bladder cancer. According to WHO, risk to health fry these by-products are extremely low compared to the risk associated with inadequate disinfection. Disinfection is common procedure for the treatment of water, the public supply. Disinfection of water involves the use of reactive chemical agents such as chlorine. Chlorine is powerful disinfection used in municipal water supplies, swimming pools and food industries. In sufficient doses, it kills the microorganisms in 30 minutes. It is also relatively cheap, readily available and remains stable in water at general storage temperature. Its antimicrobial activities are due to its oxidizing affects. The amount of free residual chlorine in treated water represent the safety factor against the pathogens surviving the actual treatment period and those which are contaminated after the treatment. When chlorination is done in municipal water supplies, its dose must be enough to leave residual free chlorine at a concentration of 0.2-0.5 mg/litters. Available chlorine may react with the dissolved or suspended organic matters, ammonia, reduced ions, manganese and sulphur compounds. So, their increased quantity lowers the chance of availability of residual chlorine. Note:

• A dose of 1 ppm destroys most of germs.

• Dose of 0.5-1.5 ppm for surface water.

• Dose of 0.1-0.2 ppm for underground water

*Processed drinking water: Nepal standard for mineral water, Residual Chlorine = <0.2 mg/l.




44

PRINCIPLE: Chlorine is primarily added to the water for destroying the harmful microorganisms. Presence of excess chlorine intensifies the taste and odours of many other compounds such as phenols etc. It may also be harmful to many aquatic organisms in combination with ammonia. Chlorine is a strong oxidizing agent and liberated iodine from potassium iodine. The liberated iodine is equivalent to the amount of chlorine and can be titrated against sodium thiosulfate using starch as indicator. The reaction preferably carried out at pH 3-4. The minimum detectable concentration using a 500 ml sample and 0.01 N sodium thiosulfate is approximately 0.04 mg of chlorine per litre. Reaction involves:

Cl2 + KI KCl + I2

I2 + Na2S4O6 NaI + Na2S4O6 REQUIREMENTS:

1. Acetic concentrated (glacial) 2. Potassium Iodide crystals 3. Sodium thiosulfate (0.025) 4. Starch solutions 5. Water samples

METHODS:

1. Take 100 ml of water sample in a conical flask and add 5ml acetic acid. The pH after addition of acetic acid should be between 3 to 4.

2. Add approximately 1 gm of Potassium iodide crystals and mix thoroughly with a stirring rod for about 15 minutes keeping it away from the direct sunlight.

3. Add a few drops of starch indicator and titrate against 0.025 N sodium thiosulfate until the content turn colourless from blue.

4. The reading should be noted. CALCULATION:

Residual chlorine (mg/L) =𝒎𝒍 ×𝑵 𝒐𝒇 𝒕𝒊𝒕𝒓𝒂𝒏𝒕 ×𝟑𝟓.𝟓 ×𝟏𝟎𝟎



S. No.

Sample Vol. Of Sample

(ml)

Burette Reading (Vol. Of sodium thiosulfate in ml)

Concurrent Cl2 (mg/ l)

Initial Final Difference

1.

2.

3.

4.




45





46

CONCLUSION:

PRECAUTIONS:

REFERENCES:




47

EXPERIMENT NO: 10

Date: DETERMINATION OF CHEMICAL OXYGEN DEMAND (COD) FROM GIVEN WATER SAMPLE OBJECTIVES:

1. To determine chemical oxygen demand in given water sample

INTRODUCTION: Water is the most vital resource for all kinds of life on this planet. According to the sources, the water environment can be subdivided as fresh water and marine water environments. Water quality can be characterized on the basis of various physical, chemical and biological parameters. A regular monitoring of many of these parameters not only prevents diseases and hazards but also checks the water resources from going further polluted. COD is amount of oxygen required for oxidation of organic waste by strong chemical oxidants. Unlike BOD, COD is not affected by toxins and other unfavorable condition for microbial growth. Its poor measure of O2 requirement for organic oxidation because O2 can be utilized for oxidation of inorganic constituent such as S, Fe, NO2 etc. Some organic matter such as C6H6

(benzene), pyrine can’t be oxidized by chemical oxidants (even strong chemical oxidants like K2Cr2O7). COD value can’t distinguish between biodegradable from non-biodegradable inorganic matter. The value of COD is always higher than BOD for same sample as COD is measure total oxidizable waste (organic + inorganic). COD test is useful for measuring O2 requirement of industrial waste and for the H2O whose BOD value can’t be measured due to presence of toxins. PRINCIPLE: COD is the measure of oxygen consumed during the oxidation of the oxidizable organic matter by a strong oxidizing agent. Potassium dichromate in the presence of sulfuric acid is generally used as an oxidizing agent in determination of COD. The sample is refluxed with K2Cr2O7 and H2SO4 in presence of mercuric sulphate to neutralize the effect of chlorides, and silver sulphate (catalyst). The excess of potassium dichromate is titrated against ferrous ammonium sulphate using ferroin as an indicator. The amount of K2Cr2O7 used is proportional to the oxidizable organic matter present in the sample.

COD, mg/l = (BA) ×N of ferrous ammonium sulphate 1000 × 8 / ml of sample

Where, A = ml of titrant with sample, B = ml of titrant with blank

REQUIREMENTS:

1. Potassium dichromate solution, 0.25N: (Dissolved 12.259 gram of dried A.R. grade

K2Cr2O7 in distilled water to make 1 litre of Potassium dichromate solution (0.025)

Dilute 0.25N K2Cr2O7 10 times).




48

2. Ferrous ammonium sulfate, 0.1N: (Dissolved 39.2 g of Fe (NH4)2(SO4)2.6H2O in water

adding 20 ml conc. H2SO4 to make 1 litre of solution. Standardize this solution with

K2Cr2O7. For standardization, dilute 10.0ml of K2Cr2O7 to about 100 ml, add 30 ml of

conc. H2SO4 and titrate with ferrous ammonium sulfate using ferroin as an indicator.

3. Ferrous ammonium sulfate, 0.01N: (Dissolve 0.1N ferrous ammonium sulfate to 10

times).

4. Ferroin indicator: (Dilute 1.485g of 1, 10-phenonitroline and 0.695g of ferrous sulfate

(FeSO4.7H2O) in distilled water to make 100ml of solution).

5. Mercuric sulfate (HgSO4, solid), sulfuric acid (conc. H2SO4, sp.gr. 1.84), silver sulfate

(Ag2SO4, solid).

6. COD flask, pipettes, hot plate or water bath

METHODS: 1. 20 ml of sample was taken in a COD flask (capacity of COD flask must be 250-500ml)

2. If the suspected COD is more than 50 mg/l, 10 ml of 0.25N potassium dichromate

solution was added. If the suspected COD is below 50mg/l, 10 ml of o0.025N

potassium dichromate solution was added.

3. A pinch of Ag2SO4, and HgSO4 was added. If the sample contains chlorides in higher

amount, HgSO4 was added in the ratio of 10:1, to the chlorides. COD could not be

determined accurately if the contains more than 2000mg/l of chlorides.

4. 30 ml of sulfuric acid was added.

5. Reflux at least for 2 hours on water bath or a hot plate. Removed the flasks, cooled

and distilled water was added to make the final volume to about 140ml.

6. 2-3 drops of ferrion indicator was added, mixed thoroughly and titrated with 0.1N

ferrous ammonium sulphate (with 0.01N ferrous of 0.025N K2Cr2O7 has been used).

7. Run a blank with distilled water using same quantity of the chemicals.

8. Then COD was calculated by using the formula.

CALCULATION:

COD, mg/L = (BA) × N of ferrous ammonium sulphate 1000 × 8 / ml of sample Where, A = ml of titrant with sample,

B = ml of titrant with blank




49


S. No Vol of Sample (ml)

Vol. of titrant consumed with sample (a)

Vol. of titrant consume with blank (b)

COD (mg/L)

1

2

3

4

5





50

CONCLUSION:

PRECAUTIONS:

REFERENCES:




51

EXPERIMENT NO: 11

Date: DETERMINATION OF IRON CONCENTRATION IN GIVEN WATER SAMPLE OBJECTIVES:

1. To determine iron concentration in water sample INTRODUCTION: Iron is one of the most abundant elements of rocks and soil ranking fourth by weight. All kinds of water including ground water have appreciable quantities of iron. Iron has more solubility at acidic pH therefore, large quantities of iron are leached out from the soil by acidic water (e.g.: acid mine drainage). Iron occurs in two valence forms iron II; ferrous and iron III; ferric. Reduce iron is generally more soluble than oxidized iron. In ground water most of iron remains in ferrous state due to general lack of oxygen. In alkaline conditions in ground waters, the iron is mostly ferrous bicarbonate which is colourless substances. When the groundwater with higher concentration of iron is trapped, it quickly oxidizes to ferric state in the form of insoluble ferric hydroxide, brown substances. If appreciable quantities of ferrous bicarbonates are present in ground water and they come in contact with oxygen at surface, the hydroxides are formed, carbon dioxide is released and hence, it increases the pH, facilitating the oxidation process.

The interconversion of iron and manganese both in their reduced and oxidized forms have a significant bearing on water chemistry with the precipitation as ferrous sulphides. Water bodies generally have higher concentration of iron at the bottom due to the prevailing reducing conditions. In addition to the natural sources of iron, corrosion of pipes, pumps etc. can also increases its concentration in distribution systems. Iron has got a microbial significance since a few microorganisms such as Crenothrix, Leptothrix and Gallionella are able to utilize dissolved iron as an energy source and convert ferrous into ferric hydroxides. This gives a rusty appearance to the water. Colonies of these bacteria may also form a slime which cause problems in water clots, pipes, pumps and distribution system. Although iron has got little concern as health hazard but is still considered as a nuisance in excessive quantities. Iron in excess of 0.3mg/l cause staining of clothes and utensils. The higher concentration of iron is also not suitable for processing of food, beverages, ice dyeing, bleaching, and many other items. Water with higher concentration of iron is used in preparation of tea and coffee, interacts with tannins to give a black inky appearance with a metallic taste. Coffee may even become unpalatable at concentration of iron more than 1.0mg/l. Potatoes also turn black on boiling in such types of waters. Iron in higher concentration may also cause vomiting. The limits on iron in waters are based on aesthetic and taste consideration rather than its physiological effects. PRINCIPLE: iron is converted into ferrous state by boiling with HCL and hydroxylamine. The reduced iron chelates with 1,10 phenanthroline at pH 3.2 to 3.3 to form a complex of orange red colour.




52

The intensity of this colour is proportional to the concentration of iron and follows Beer’s law and therefore can be determined colorimetrically. Strong oxidizing agents cyanide, nitrite and phosphates (mainly polyphosphates) of chromium, zinc, cobalt, copper and nickel interfere with the determination of iron. The boiling of the sample with an acid initially removes interference of cyanides, nitrite and polyphosphates. Hydroxylamine eliminates the interference caused by strong oxidizing agents. Bismuth, Cadmium, Mercury and Silver precipitate the phenanthroline and the errors caused by them and other metals can be overcome by using excess of phenanthroline. REQUIREMENTS:

1. Hydrogen chloride 2. Hydroxylamine HCL solution 3. Ammonium acetate buffer solution 4. Phenanthroline solution 5. Stock iron solution (200mg Fe/L) 6. Standard iron solution (10mg Fe/L)

METHODS:

1. 50ml water sample containing not more than 4mg/l iron was taken in a 150ml conical flask.

2. 2ml conc. HCl and 1ml of hydroxylamine-HCl solution was added. 3. The contents were then boiled to half of volume for dissolution of all the iron and then

cooled. 4. 10ml ammonium acetate buffer and 2ml phenanthroline solution was then added to

it. (If the sample contains interference of heavy metals then 10ml of phenanthroline was added instead of 2ml. An orange red colour was appeared.)

5. Volume up to 100ml was made and after 10 minutes the reading was taken on a spectrophotometer at 510nm.

6. Standard curve in the range of 1 to 4mg/l of iron was prepared using various dilutions of standard iron solution.

7. IF only the soluble iron is to be determined the sample was acidified directly with 1ml conc. HCl and proceed from the step 4 onwards.

8. Then the concentration of iron was calculated directly from the standard curve. OBSERVATIONS: Table 1:

S. No Vol of water (ml)

Final Conc. of iron (mg/l) Absorbance at 510 nm (OD510)

Inference

1 0

2 1

3 2

4 3

5 4

6 Sample




53





54

CONCLUSION:

PRECAUTIONS:

REFERENCES:




55

EXPERIMENT NO: 12

Date: DETERMINATION OF ARSENIC CONCENTRATION IN GIVEN WATER SAMPLE OBJECTIVES:

1. To determine arsenic concentration in water sample INTRODUCTION: Arsenic is a crystal-shape metalloid element, brittle in nature and grey or tin-white in color. It is a common contaminant of ground water. It is present in the waste water of many industries such as ceramics, tanneries, chemical, metal preparation and pesticides. It has a tendency to get accumulated in body tissues to cause arsenosis. Arsenic may be found mainly in two forms, arsenate [As (V)] and arsenite [As (III)]. The presence of arsenic in drinking water is due to either its natural presence in surface and in ground waters, or as a result of human activities such as industrial applications, leather and wood treatments, use of pesticides. As per WHO’s recommendation, the maximum allowable contamination level of arsenic in drinking water is 10 µg/L. It affects liver and heart, and is also reported to be carcinogenic. The arsenic contamination of ground water has been found to adversely affect the human body at a level as low as 0.01 mg/l. The maximum permissible limit in the drinking water contaminant level for arsenic as per WHO is 0.01 mg/L. Arsenic is mainly present in natural water in inorganic trivalent (AsO) and penta-valent (As oxidation states). There are different analytical techniques for inorganic arsenic detection, such as atomic absorption spectrophotometry (AAS), hydride generation-AAS, inductively coupled plasma, neutron activation analysis, an electrochemical method, colorimetry etc. Most of these techniques are expensive and require some training to handle them efficiently.

PRINCIPLE:

Arsenic, in presence of Zn in acid medium gets reduced to arsine, AsH3, Arsine is then passed through a scrubber containing glass wool soaked with lead acetate, and later is absorbed in silver diethyl-dithiocarbamate dissolved in pyridine. Arsenic reacts with the silver salt to form a red complex which can be determined colorimetrically.

Many other metals such as Cr, Co, Cu, Hg, Mo, Ni, Pi, Sb and Ag interfere in the detection of As, but the concentration of these metals normally encountered in the waters are often less to produce any significant interference.

REQUIREMENTS:

1. Arsine generator and absorber assembly




56

2. Spectrophotometer: with adjustment at 535nm 1cm cells

3. Hydrocholric acid conc

4. Potassium iodine solution: Dissolve 15g KL in distilled water to prepare 100ml of solution. Store in a brown bottle.

5. Stannous chloride solution: Dissolve 40g SnCl2.2H20 in 100ml conc. HCL

6. Lead acetate solution: Dissolve 10g Pb (C2H302)2.3H20 in distilled water to prepare 100ml of solution.

7. Silver diethyl-dithiocarbamate reagent (SDDC): Dissolve 1gm AgSCSN in pyridine to prepare 200ml of the reagent. Store in a brown bottle.

8. Zinc: Arsenic free, 20 to 30 mesh.

9. Standard arsenic solution (1ml=1µg As): Dissolve 1,320g arsenic trioxide, As2O3 in 10ml distilled water having 4g NaOH and make up the volume to 1litre.This is 1000mg As/L stock solution. Dilute this stock solution 100 times. This is arsenic solution containing 10meu g As in 1ml.Dilute this solution further 10 times, to prepare standard solution of As.

METHODS:

1. Take 35ml sample into the arsine generator and add in succession, 5ml conc. HCl, 2ml KI solution, and 0.4ml (8 drops) SnCl2 reagent, thoroughly mixing the sample after each addition. Keep for about 15minutes.

2. Soak the glass wool in scrubber with lead, acetate solution taking care that the solution should not drain into the generator.

3. Take 4ml silver diethyl-dithiocarbamate reagent in the absorber tube.

4. Add 3g Zn in the generator and immediately connect the assembly with all the joints air tight.

5. Keep for about 30min for the generation of arsine with the slight heating of the generator. The gas will be absorbed in the SDDC reagent.

6. Remove solution from the absorber and measure the intensity of the color at the 535nm using the reagent blank as a reference.

7. Prepare the standard curve in the range of 0.0 to 10.0g. As by taking suitable volumes of the standard as solution and following the same procedure as for the sample.

CALCULATION:

𝑨𝒔, 𝒎𝒈/𝑳 =𝒈 𝑨𝒔





57


S. No. Sampling sites





58

CONCLUSION:

PRECAUTIONS:

REFERENCES:




59


MICROBIAL QUALITY ANALYSIS OF WATER ESTIMATION OF COLIFORM DENSITY IN WATER SAMPLES BY MPN METHOD OBJECTIVES:

1. To estimate coliform density in the water sample by Most Probable Number (MPN) method

INTRODUCTION:

Coliforms: A coliform is a group of non-spore-forming, facultatively anaerobic, gram-negative

rod, which ferments lactose to acid and gas within 48 hours at 35°C. Coliforms are almost

always Enterobacteriaceae from the genera Enterobacter, Klebsiella, and Escherichia (also

lactose positive strains of Citrobacter). Klebsiella are of interest due to the ability of most

strains to use N2 as sole nitrogen source. Study of this organism has helped to unlock many

of the mysteries of nitrogen fixation (an agronomically important process). K. pneumoniae is

a human pathogen sometimes causing pneumonia. Enterobacter and Klebsiella are widely

distributed in water, soil, and plant material. One member of the genus Escherichia, E. coli, is

the star organism of biology. This organism's main habitat is the intestinal tract of warm

blooded animals, but it can also be found in environments contaminated with feces. Some

strains of E. coli also cause gastrointestinal disease. Several recent severe outbreaks have

been traced to undercooked meat infected with pathogenic E. coli.

The experiment involves the analysis of water for fecal contamination. Enteric organisms play an important role in this process.

Water Analysis: Water, the universal solvent, is essential to all life. Critical to our modern

civilization is the availability of a clean water supply for bathing, drinking and cooking.

Unfortunately, many pathogens are transmitted through the water supply. Some of these

disease-causing pests enter water from the feces of ill individuals and are then ingested and

thereby transmitted to others. Diseases such as polio, typhoid, cholera, hepatitis, shigellosis,

salmonellosis and others can spread in this manner. To assure a safe water supply, it is critical

to monitor for the presence of these pathogens. However, it would be expensive and time

consuming to check the water supply for all of them; instead, an indicator organism is used

to assay for fecal contamination. Indicator organisms must have four properties to be useful

for water analysis.

1. The only natural environment of the microbe should be in association with feces and it should always be present.

2. It should not grow outside of its natural environment.




60

3. The bacterium should survive longer than the most viable pathogen, but not so long so that historical events are detected.

4. It should be easy to detect.

Coliforms come closest to fulfilling all these criteria and are the standard indicator organisms used to test for the biological pollution of water. Enterobacter and Klebsiella are able to survive and multiply in the environment and are therefore not the best indicators of fecal pollution. The sole habitat of E. coli and K. pneumoniae, termed fecal coliforms, is the intestines of warm blooded animals. Thus, fecal coliforms are good indicators of fecal pollution and can be differentiated from other coliforms by incubating on selective media at 44.5°C.

MPN technique is most commonly applied for quality testing of water i.e. to ensure whether the water is safe or not in terms of bacteria present in it. This technique is mainly used for coliforms count and also can be used for faecal streptococci, Vibrio cholerae, Salmonella and some other pathogens. The number of organisms present in sample is reviled by standard MPN charts in terms of number of organisms/100 ml of sample. Water to be tested is diluted serially and inoculated in lactose broth, coliform if present in water utilize the lactose present in the medium to produce acid and gas. The presence of acid is indicated by 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 coliform is determined by counting the number of tubes giving positive reactions (i.e. both color change and gas production) and comparing the pattern of positive results with standard statistical tables. MPN test is performed in 3 steps 1. Presumptive test: The presumptive test, is a screening test to sample water for the presence of coliform organisms. Presumptive test is enrichment technique in which either lactose broth or lauryl sulphate broth is used. Tryptose lauryl broth can also be used. Commonly used medium is Mac–Conkey broth that contain indicator bromo-cresol purple. 3 sets or tubes, 1 set of double strength and 2 sets of single strength broth are first prepared. An inverted Durham tube is placed in each tube of medium without any air bubbles. Then all media within test tubes are cotton plugged and autoclave. After autoclaving the media is sufficiently cooled. And required amount of sample is added in each media and after that incubated at 35.50C for 24 hrs. If presumptive test is negative, no further testing is performed and water source considered microbiologically safe. If, however, any tube in the series shows acid and gas, the water considers unsafe and the confirmed test is performed on the tube displaying a positive reaction. The method of presumptive test varies for treated and untreated water. 2. Confirmatory test: The test is performed to confirm the positive result of presumptive test. The media used in this technique is BGLB 2 % broth (Brilliant Green Lactose Bile Broth). The brilliant green and bile inhibit the Gram-positive bacteria such as Lactobacillus, Streptococcus Bacillus and




61

Clostridium. 0.013 gm/lit media concentration is tolerated only by coliform but not by gram negative bacteria. Hence the gas production is only by coliform. Here one loopful of organism from each positive tube from presumptive test is inoculated in each respective tube containing BGLB broth 2% and incubated at 35.50C for 24 hrs. Gas production in Durham’s test tube is observed. The result may be positive and doubtful. Again, incubating for 24 hrs. the result is interpreted finally.10% or more gas production is taken as positive and less than 10% is taken as negative.

Coliform density/100 ml = MPN/100 ml

=𝑵𝒐. 𝒐𝒇 𝒑𝒐𝒔𝒊𝒕𝒊𝒗𝒆 𝒕𝒖𝒃𝒆𝒔 𝒙 𝟏𝟎𝟎

√𝑽𝒐𝒍. 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 𝒊𝒏 𝒏𝒆𝒈𝒂𝒕𝒊𝒗𝒆 𝒕𝒖𝒃𝒆𝒔 𝒙 𝑻𝒐𝒕𝒂𝒍 𝒗𝒐𝒍. 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 𝒊𝒏 𝑬𝒙𝒑𝒆𝒓𝒊𝒎𝒆𝒏𝒕

3. Completed test: This is the final test in which a loopful of sample from confirmatory test is streaked onto two selective media like EMB or M-endo agar, Agar slant and finally again in lactose broth. Firstly, it is streaked on EMB or M-endo agar plates are incubated at 370C for 24 hours and observed for typical, atypical and non-typical colonies. Typical colonies: These are pink colonies with dark color having greenish metallic sheen or nucleated sheen. Generally, E. coli form typical colonies. Atypical colonies: These are pink colonies with non-nucleated colony formed by coliforms or lactose fermenting non-coliforms. E.g. Bacillus spp, Lactobacillus spp. Non-typical colonies: these are non-pink colonies formed by non-coliforms. The typical and non-typical colonies are then transferred into agar slant and again transferred in lactose broth. The gas production in the broth finally confirms the organism to be coliforms. Also, there is necessary to determine that the coliform is of faecal origin or not. For this, the culture from positive test are inoculated into EMB agar and incubated at 44.50C for 24 hrs. REQUIREMENTS:

1. Medium: Lactose broth or Mac-Conkey broth or Lauryl tryptose broth, EMB

2. Glassware: Test tubes of various capacities (10ml, 1ml, 0.1ml), Durham tube

3. Others: Sterile pipettes

METHODS:

A. Presumptive test:

1. 3 double strength lactose broth, 3 single strength lactose broth & 3 single strength lactose broth tubes were labeled as 10ml, 1ml & o.1ml respectively.

2. Then, 10ml of sample to each DSLB tube, 1ml of sample to each middle set of SSLB tube and 0.1ml of sample to each SSLB tube was transferred aseptically with the help of 10ml, 1ml, and 0.1ml pipettes respectively.

3. All tubes were incubated at 35.50C for 24 hours.




62

B. Confirmatory test:

1. One loopful of sample from each of positive lactose broth tubes were taken & inoculated in BGLB broth.

2. All tubes were incubated at 370C for 48 hrs.

C. Complete test:

1. One loopful sample from each of the positive BGLB tubes were taken.

2. Then, sample or inoculum was streaked into various Eosin methylene blue agar plates with the help of sterile inoculating loop.

3. Plates were incubated at 370C and 44.50C for 24 hrs. and result was observed.

OBSERVATIONS:

Table 1: For Presumptive test

Samples No. of tubes giving positive results MPN index per

100ml

95% confidence limits

10ml tubes 1ml tubes 0.1ml tubes Lower Upper

Table 2: For Confirmatory test

Presumptive sets No. of positive or doubtful tubes

No. of positive in BGLB broth (> 10% gas production)

Inference

10ml tubes

1ml tubes

0.1ml tubes

Table 3: For completed test

Sample Technique Media Colony Characteristics Inference

Color Shape Elevation

EMB / M=endo




63

Table 4: Biochemical tests

S. No. Tests Performed Tests result Inference

1 IMViC test

2 OF test

3 TSI Agar test

4 Urease test





64

CONCLUSION:

PRECAUTIONS:

REFERENCES:




65

EXPERIMENT NO: 13 Date: ESTIMATION OF COLIFORM DENSITY IN WATER SAMPLES BY MF METHOD OBJECTIVES:

1. To estimate coliform density in the water sample by Membrane Filter (MF) method INTRODUCTION:

The Membrane Filter (MF) procedure was introduced to bacteriological water analysis in 1951, after its capacity to produce results equivalent to those obtained by the MPN procedure was demonstrated (Clark et al., 1951; Goetz and Tsuneishi, 1951). It is a quantitative procedure that uses membrane filters (Nitrocellulose, Nitroacetate, Nitrolactate, etc) with pore sizes (most commonly of 0.45 microns) sufficient to retain the target organisms. The water sample is filtered through the membrane, which is then transferred to an appropriate selective growth medium for identification and quantitation. This procedure has the advantages of being able to examine larger volumes of water than with MPN, as well as having an increased sensitivity and reliability and requiring significantly reduced time (1 day vs. 3 to 4 days), labour, equipment, space, and materials. These qualities have made the MF technique the method of choice in some jurisdictions for the routine enumeration of coliforms in drinking water. One disadvantage of the MF procedure that should be noted is that it cannot be used on highly turbid water samples. The particulate matter concentrated by the filter can interfere with colony development and with the production of surface sheens used for visual detection of coliforms. Besides metals and phenols can stick to the filter inhibiting growth, and non-coliforms in the test sample may interfere with the formation of coliform colonies on the plate.

Unlike the MPN procedure that employs liquid medium in test tubes and the sample of drinking water being add to the test tubes. Membrane filter procedure employs the different technique. In the common membrane filter procedure, the sample of water to be tested is passed through a sterile membrane filter, which removes the bacteria and the filter is placed on a culture medium in petri dish. Special petri dishes of a size accommodate the filter disk are employed for incubation. The sample of at least 100ml should be filtered at a single test but it depends upon the microbial load or nature of pollution. Membrane filter is generally composed of cellulose ester having pore size ≈ 45µm and about 80 of membrane filter is occupied with these pores. Upon incubation colonies will develop upon the filter disk wherever bacteria were entrapped during the filtration process. PRINCIPLE: Microbial cell numbers are frequently determined using special membrane filters processing millipores small enough to trap bacteria. In this technique a water sample containing microbial cells passed through the filter. The filter is then placed on a pad socked with nutrient broth and incubated until each cell develops into a separate colony. Membranes with different pores sizes are used to trap different microorganisms. Incubation times for membrane also very with medium and the microorganism. A colony count gives the number




66

of microorganism in the filtered sample, and specific can be used to select for specific microorganisms. This technique especially useful in analyzing aquatic sample. REQUIREMENTS:

1. Samples 2. Filtrations apparatus 3. Petri dish 4. Vacuum pump 5. Absorbent pad 6. Membrane filter 7. Forceps

METHODS: A. Confirmatory / Completed test:

1. Collect the sample and make any necessary dilutions. 2. Select the appropriate nutrient or culture medium, dispense the broth into a sterile

petri dish, evenly saturating the absorbent pad. 3. Flame the forceps and remove the membrane from the sterile package 4. Place the membrane filter into the funnel assembly. 5. Flame the pouring lip of the sample container in pour the sample into the funnel. 6. Turn on vaccum and allow the sample to draw completely through the filter. 7. Rise funnel with sterile buffered water. Turn on vaccum and allow the liquid to draw

completely through the filter. 8. Flame the forceps and remove the membrane filter from the funnel. 9. Place the membrane filter into the prepared petri dish. 10. Incubated at the proper temperature and for the appropriate time period. 11. Count and confirm the colonies and report the results.

B. Confirmatory / Completed test:

1. Transfer typical colonies or colonies with metallic sheen to lactose broth. 2. Incubate the broth at 37°C for 24 hrs. and observe for gas production. 3. Further, perform subculture, Gram staining and Biochemical tests for strain

identification. OBSERVATIONS: Table 1:

S. No. Samples Volume of sample

Media used

Types of colony

cfu/100ml Inference




67





68

CONCLUSION:

PRECAUTIONS:

REFERENCES:




69

EXPERIMENT NO: 14 Date: MICROBIOLOGICAL ANALYSIS OF AIR SAMPLES BY GRAVITY SEDIMENTATION METHOD OR SETTLING PLATE TECHNIQUE OBJECTIVES:

1. To isolate, enumerate and identify microbes in air sample by gravity sedimentation method or setting plate technique in indoor

INTRODUCTION:

Air is not a natural environment for the growth and reproduction of microorganisms. It doesn’t contain the necessary amount of moisture and utilizable form of nutrients. Yet microorganisms are found in air, though they have a transient survival. The microorganisms come from soil, from organic waste of man and animals during their various activities. The kinds and numbers of microorganism in the air vary, depending upon the sources of contamination in the environment, locality, atmospheric conditions e.g. speed of the air current, humidity, sunlight, temperature, size of particles on which they are attached and nature of microorganisms. The miroflora of air consists chiefly of spores of fungi, especially Penicillium, Aspergillus, Cladosporium, Homodendrum, Alternaria, ascospores of yeasts, conidia of Streptomycetes, and endospores of Bacillus and Clostridium. Among the non-spore forming bacteria, organisms belonging to the genera Sarcina, Micrococcus, Alcaligens, Chromobacterium, etc. are also found in air. There are several methods of microbial analysis in air. One of the simple techniques is settling-plate technique which uses the application of gravity. In this method the cover of the petridish containing agar medium is opened at specific site and the agar surface is exposed to the air keeping it at certain height above the ground for several minutes. On incubation, a certain number of colonies develop which can be then analyzed for further identification. REQUIREMENTS:

1. Petridish containing agar media 2. All reagents and media for identification 3. Microscope / loop / glass rod

METHODS:

1. Take sterilized agar media to the sites for sampling (don’t open until it is being exposed).

2. Expose the agar plates at specific sites removing caps for 10 minutes. 3. Again, close the cap of petridish and bring to lab safely without any contamination. 4. Incubate the plates at 28-30 °C (environmental temperature) for 24 hrs (48-72 hrs for

fungi). 5. After incubation, observe for colonies development and enumerate the colonies. 6. Purify each colony by repeated sub-culturing and perform standard procedure for

identification of different microbes’ present.




70

7. Evaluate the microbial condition of respective environments. OBSERVATIONS: Table 1:

S. No. Sampling sites

Media used

No. of microbes

Types of microbes Inference





71

CONCLUSION:

PRECAUTIONS:

REFERENCES:




72

APPENDIX-I

Water quality

parameters

Units Nepal’s

NDWQS*

WHO WaterAid

standards

for Nepal

Selection criteria and rationale

Electrical

conductivi

ty

lS /cm 1500

TDS mg/L 1000 1000 500 (1000) Above this, the water may become consumer

unacceptable and may cause gastro-intestinal

irritation. Color TCU 5 (15) 15 5 (15) Acceptability to users; Colour above 15 TCU can be

detected in a glass of water so above this

consumer may not accept the appearance

Ph

ysic

al Turbidity NTU 5 (10) 5 ≤ 5 • High levels of turbidity can stimulate bacterial

growth and protect micro-organisms from

the effects of disinfection.

• Above 5, opalescence increases and consumer

acceptability decreases; For effective terminal

disinfecting, more disinfectant is consumed

Odor

and

taste

Non-

objecti

onable

Non-

objecti

onable

Non- objectionable

Consumer acceptability

C

he

mic

al p

ara

me

ters

pH

Total hardness

mg/L 500 - 300 (500) Above this encrustation inside supply lines may occur

depending upon other water characteristics such as

pH, Alkalinity, Total dissolved solid etc. and may

produce adverse effect in domestic uses.

Nitrate (NO3)

mg/L 50 50 50 Above this value, Methemoglobinemia (Blue-Baby

Syndrome) take place.

Iron mg/L 0.3 (3) 0.3 0.3 (3) Above this imparts taste and colour; effects on

domestic uses, water supply structures in absence of

alternative sources the value may be extended up to

3

Manganese mg/L 0.2

Arsenic (As) mg/L 0.05 0.01 0.01 (0.05) • For new water points, value should be

within 0.01 and for old water points, value

up to 0.05 is acceptable.

• As a precautionary measure any new tube

wells installed by WAN should show no

arsenic presence when tested with a field

test kit at installation

• For new tube wells, 0.01 is adopted provided

PeCO75 field test kit or lab testing by AAS is

used.

• Also Refer to WAN’s Arsenic Testing Protocols

Ammonia (NH3)

mg/L 1.5 1.5 1.5 National Water Quality standard also

recommended the same value as that of WHO

guideline value.

Chloride mg/L 250 250

Fluoride(F) mg/L 0.5 (1.5) 1.5




73

Free

(Residual)

Chlorine

mg/L 0.1-0.2 - 0.2 (0.5) • Excess residual chlorine may produce

harmful disinfectant by- products etc.

Residual chlorine level may be used in rapid

assessment of bacterial quality

• To be applicable when water is

chlorinated. Zinc mg/L 3 3 3 • Impacts a bitter taste at concentration

above this value. Value is relevant only for

RWH and thus recommended to test before

scaling up

• The WHO does not have any health- ba s e d

limits. (Refer WHO - Vol:2)

M

icro

bio

log

ica

l (B

act

eri

olo

gic

al)

pa

ram

ete

rs

Thermotol

erant

Faecal

Coliform

(E. coli)

MPN/

100ml

0 nil 0/100 ml. (No Risk)

• The WHO Guideline Value in the 2nd edition of

Guidelines for Drinking Water Quality for

thermotolerant coliforms (often referred to as

faecal coliforms) is 0cfu/100ml, but Guidelines

explicitly state that a relaxation of up to 10

fc/100ml is acceptable in community-

managed un-chlorinated supplies.

• WEDC has also recommended the limit of

10fc/100ml which is classified as “low risk”

to health by WHO.

• For most water supply schemes in remote areas

of Nepal, chlorination of water supply is not

possible due to inaccessible geographical

location. In this context, For WAN and its

partners, Water quality standards has been if

according to degree of contamination and

urgency for action.

1-10/100 ml. (Low Risk)

11-100/100 ml. (High Risk)

101-1000 (Very High Risk) Total

coliform

MPN/

100ml

0 in 95%

samples

nil 0 in 95 % samples




74

APPENDIX II

THE MPN TABLE FOR 3 SETS 3 TUBES METHOD

Number of positive tubes in

MPN / 100 ml

Number of positive tubes in MPN / 100 ml

Set I (10 ml)

Set II (1 ml)

Set III (0.1 ml)

Set I (10 ml)

Set II (1 ml)

Set III (0.1 ml)

0 0 0 <3 2 0 0 9.1

0 0 1 3 2 0 1 14

0 0 2 6 2 0 2 20

0 0 3 9 2 0 3 26

0 1 0 3 2 1 0 15

0 1 1 6.1 2 1 1 20

0 1 2 9.2 2 1 2 27

0 1 3 1.2 2 1 3 34

0 2 0 6.2 2 2 0 21

0 2 1 9.3 2 2 1 28

0 2 2 12 2 2 2 35

0 2 3 16 2 2 3 42

0 3 0 9.4 2 3 0 29

0 3 1 13 2 3 1 36

0 3 2 16 2 3 2 44

0 3 3 19 2 3 3 53

1 0 0 3.6 3 0 0 23

1 0 1 7.2 3 0 1 39

1 0 2 11 3 0 2 64

1 0 3 15 3 0 3 95

1 1 0 7.3 3 1 0 43

1 1 1 11 3 1 1 75

1 1 2 15 3 1 2 120

1 1 3 19 3 1 3 160

1 2 0 11 3 2 0 93

1 2 1 15 3 2 1 150

1 2 2 50 3 2 2 210

1 2 3 24 3 2 3 290




75

APPENDIX III

Photographs relating to MPN test

Photograph 1: Presumptive test (Lactose Broth)




76

Photograph 2: Confirmatory test (2% BGLB Broth)

Photograph 3: Completed test (EMB-metallic sheen and M-endo Agar-fish eye)




77

BIBLIOGRAPHY

APHA 1999, Clesceri L.S. Greenberg A. E. and Eaton A. D. 1999 Standard Methods for the

Examination of Water and Wastewater, American Public Health Association, American

Water Works Association, Water Environment Federation, 20th Edition.

Benson. 2001 Microbiological Applications Laboratory Manual in General Microbiology,

Eighth Edition, the McGraw-Hill Companies.

Cheesbrough M. 1984 Medical Laboratory Manual for tropical countries Volume II ELBS

publishing.

Goel P.K. 2001 Water Pollution Causes Effects and Control New Age International Publishers.

Nepal’s National Drinking Water Quality Standards (2006).

Trivedy R. K. and Goel P.K. 1986 Chemical and Biological Methods for Water Pollution studies.

Environmental publications, India.

WHO’s ‘Guidelines for Drinking Water Quality’ (2004).

http://www.studentsguide.in/Microbiology

http://www.studentsguide.in/Microbiology

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