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Laboratory pipettes are classified by their method of liquid transfer. There are two types of pipette one is plastic which is made up of polystyrene and are essentially disposable and second one is glass pipettes are very resistant to chemicals. Glass pipette can be used frequently by washing, sterilising and dry heat sterilisation is also available. Some of the common types of pipettes are using in laboratory such as serological, volumetric, micropipettes, multichannel, and Pasteur pipettes. Serological and graduated pipettes are used for transferring various volumes, while micropipettes and volumetric types of pipettes are only considered for precise measurement and transfer. Multichannel pipettes are used for dispensing into multiple wells, and Pasteur pipettes are simple for transferring small amounts.
1. Serological Pipettes
• Also known as graduated pipettes, made of glass or transparent polystyrene they are calibrated with markings to measure and dispense varying volumes.
• Appropriate for cell culture applications, transferring liquids and mixing.
2. Multichannel Pipettes
• It is design to transfer multiple samples at once, often used for 96 well plates or pcr tubes.
• It can be manual or automated, with multiple heads for faster dispensing.
3. Volumetric Pipettes
• It is designed to supply for single and particular volume of liquid.
• Marked by single graduation for an exact volume, such as 1, 5, 10, 25, and 50 mL.
• Used in chemical reactions and analysis where accuracy is crucial.

The total plate count (TPC) is also called the Bacterial plate count (BPC), aerobic plate count, standard plate count (SPC), total viable count (TVC) or mesophilic count represents the number of colony forming units (CFU) per g (or per mL) of growing microorganisms such as bacteria, yeast and mold on a nonspecific solid bacteriological growth medium under the specified conditions. Temperature, incubating time, medium type, and any other conditions used in the enumeration varies upon reference standards and laboratory objectives. Laboratories participating in the program choose their own routine conditions.
Principle
Usually direct count of too many bacteria in a sample is difficult but the bacterial colonies may be used as a measure of the bacterial cells in the known serially dilution and plated out on an agar surface in such a way that single isolated bacteria form visible isolated colonies. However, colony-forming unit might actually consist of a chain of bacteria rather than a single bacterium if the organism often develops various cell configurations, such chains. Furthermore, a portion of the bacteria might be grouped together. Thus, typically refer to the process of using the plate count technique as a means of estimating the number of colony forming units (CFUs) in that known solution. Then further this figure use to extrapolate the total amount of CFUs present in the original sample.

Sulfate can have harmful effects on aquatic life in freshwaters, sulfate cause osmotic stress or specific ion toxicity in aquatic organisms, especially in soft waters where Ca2+ and Mg2+ concentrations are low. Egg fertilization and early embryonic development were the most sensitive developmental stages of whitefish to sulfate (Karjalainen et al., 2021).
Sulphate (SO4 2-) may be estimated by either turbidimetric or gravimetric methods (APHA, 1995)
1. Turbidimetric method is more applicable in the range of 1- 40 mg Sulphate (SO4 2-)/ L
2. Gravimetric method is suitable if the concentration of Sulphate (SO4 2-) is above 10 mg/ L
Turbidimetric Method (Range 1 - 40 mg/L of sulphate) Principle
Sulphate ion (SO4 2-) precipitated with barium chloride (BaCl2) in an acetic acid medium in order to form crystals of barium sulphate (BaSO4).
BaCl2 + SO4 2-+ BaSO4 (precipitate)
The formation of these crystals is increased in the presence of an acetic acid solution containing sodium acetate, potassium nitrate, acetic acid and magnesium chloride. The suspended barium sulphate (BaSO4) is measured by spectrophotometer and concentration of sulphate (SO4 2-) is determined by comparison of the reading with a standard curve. Minimum detectable concentration is approximately 1 mg (SO4 2-) / L.

Phosphorus is nearly exclusive found as phosphates in waters and wastewater and it categorised in to: a) orthophosphate, (b) organically bound phosphate and (c) condensed phosphates. Theses all three forms of phosphorus occur in solution, in the body of aquatic organisms through food chain. All the forms of phosphorous, whether dissolved or particulate are converted to inorganic forms (phosphate) after digestion of sample. Various methods use for the digestion of the sample including use of perchloric acid, H2SO4- K2SO4, H2SO4- HNO3 etc. The H2SO4- HNO3 digestion technique is discussed here.
Reagents
1. Phenolphthalein indicator
2. Conc. sulphuric acid (H2SO4)
3. Conc. nitric acid (HNO3)
4. Sodium hydroxide (NaOH), 1 N: Dissolve take 40 g of NaOH in 1000 ml of distilled water
Procedure
Digestion
1. Take 50 ml of sample in a Kjeldahl flask
2. Add 1 ml H2SO4 and 5 ml HNO3
3. Once the HNO completely removed, digest the sample on a hot plate until the volume approaches 5 ml. Then, keep heating the solution until it loses colour (you may also heat it in a beaker covered with a watch glass to prevent excessive evaporation).

The most oxidizable form of nitrogen is nitrate nitrogen (NO3 -N), which is found in minimal amounts in surface waters but can reach considerable concentrations in some aquifers. An essential plant nutrient, nitrate also contributes to eutrophication in receiving water bodies.
Principle
Nitrate reacts with phenol disulphonic acid and produces a nitro-derivative which in alkaline solution develops yellow colour due to rearrangement of its structure. The yellow colour follows Beer’s law and is proportional to the concentration of nitrate present in the sample. Ultraviolet (UV) technique measures the absorbance of nitrate at 220 nm, which is suitable for screening uncontaminated water (low in organic matter). A second measurement made at 275 nm, may be used to correct the nitrate value (because 275 nm is not absorbed by nitrate, but absorbed by other matter). The nitrate calibration curve follows Beer’s law up to 11mg NO3 -N /L.
Reagents
1. Stock nitrate solution (1ml=100 µg NO3 -N): Dry potassium nitrate (KNO3) in an oven at 105 oC for 24 h. Take 360.9 mg potassium nitrate and dissolve in distilled water and dilute to 500 ml. Preserve it with adding 1 ml of chloroform in per 500 ml stock nitrate solution. This solution will stable for at least 6 months.
2. Intermediate nitrate solution (1 ml = 10µg NO3 -N): Take 100 ml of stock nitrate solution and dilute it to 1000 ml of distilled water. This solution can be preserve by adding 2 ml of chloroform per 1000 ml. This Intermediate nitrate solution will stable for at least 6 months.
3. Hydrochloric acid (HCL) solution, 1N: Take 83 ml of hydrochloric acid and dilute it to 1000 ml of distilled water

Nitrite (NO2-) is formed in waters by oxidation of ammonia compounds (by aerobic nitrifying bacteria, e.g. Nitrosomonas) or by reduction of nitrate (by facultative anaerobic denitrifying bacteria, e.g. Pseudomonas). Such oxidation and reduction may occur in wastewater treatment plants, water distribution system, and natural waters. As an intermediate stage in nitrogen cycle, it is unstable. Very high nitrite levels are usually associated with waters having unsatisfactory microbiological activity.
Principle (Colorimetric method)
When nitrite (NO2 -) as nitrous acid (HNO2) mixes with sulfanilamide in an acidic environment (pH 2-2.5), a diazonium salt is created. This salt then combines with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED dihydrochloride) to make a vivid pinkish red azo-dye. The amount of nitrite in the sample directly correlates with the colour that is created. At 543 nm, the colour follows Beer’s law up to 180 µg/L with a 1 cm light path. By diluting a sample, a higher nitrite concentration can be determined. Sulfanilamide + HNO2 + HCl ? Diazonium salt + H2O.
Diazonium salt + NED-dihydrochloride ? Red colouredazo dye

Fish excrete Ammonia, is a byproduct from protein metabolism and bacteria break down organic waste such as discarded food, faeces, dead planktons, sewage, and so on. When ammonia is unionized form (NH3) is extremely dangerous; yet when ammonia ionized form (NH4 +) it is harmless; both forms are known as total ammonia. Fish suffering from ammonia toxicity often”become lethargic and frequently emerge near the surface gasping for oxygen. common effects of ammonia in the range >0.1mg/l include Gill damage, breakdown of mucous-producing membranes, “sub-lethal” effects like reduced growth, poor (FC) feed conversion, and reduced disease resistance at concentrations lower than lethal concentrations, osmoregulatory imbalance, and kidney failure.
Principle
When Ammonia react with alkaline Nessler reagent (K2HgI4 or 2KI + HgI2) it produces a yellowish-brown coloured compound. Pretreatment with ZnSO4 and NaOH precipitates Ca, Mg, Fe and sulphide, which form turbidity and apparent colour. Addition of ethylene diamine tetraacetic acid, EDTA (before nessler’s reagent) or Rochelle salt solution prevents precipitation of residual Ca and Mg in presence of the alkaline Nesslers’s reagent.

Principle
Chloride ion is determined by Mohr’s method, titration with standard silver nitrate solution in which silver chloride is precipitated first. The end of titration is indicated by formation of red silver chromate from excess AgNO3 and potassium chromate used as an indicator in neutral to slightly alkaline solution.
AgNO3 +Cl- ? AgCl+NO3
2AgNO3+ K2CrO4 ? Ag2CrO4+2KNO3 (Reddish Brown)
Reagents
1. Standard Silver nitrate 0.0141N : Take 1.189 g and dissolved it in 500 ml distilled water.
2. Sodium chloride 0.014N: Dissolve 0.28 g sodium chloride in 500 ml of distilled water.
3. Potassium Chromate indicator
Procedure
1. Take 50 ml sample in a 100 ml conical flask
2. Adjust its pH in between 7.0 and 8.0 either with alkali or acid solution. Otherwise, AgOH is formed at high pH level or CrO4 -2 at low pH level.
3. Add 1 ml of potassium chromate (K2CrO4) colour turn to light yellow.
4. Titrate with standard silver nitrate (AgNO3) solution till colour change from light yellow to brick red colour.
5. Note the volume of AgNO3 added (A)
6. For better accuracy, titrate distilled water in the same manner
7. Note the volume of silver nitrate added for distilled water (B)

Hardness is the total soluble Ca and Mg Salts (in same cases Fe salts). It includes sulphates and chlorides along with carbonate, bicarbonate and hydroxide salts. Soft water: 0-100 mg/L, Hard water: 100 - 300 mg/L, Very hard water: > 300 mg/L CaCO3. Total hardness should be 50- 300 mg/L. Very hard water causes osmoregulatory stress to fish. Hardness below 50 mg/L reduces growth of plankton.
Reagents
1. 10.01M EDTA solution: Dissolve 3.72 g disodium salt of EDTA in distilled water and make the volume 1L by shaking.
2. Eriochrome Black T (EBT) Indicator: Add 0.2 g dry Eriochrome powder to 50 ml of triethyl amine. Add 5 ml absolute alcohol and shake it.
3. Ammonium chloride-Ammonium hydroxide buffer solution: Dissolve 17.5g ammonium chloride (NH4Cl) in 142 ml of ammonia solution (NH4OH). Add distilled water to make the volume 250 ml.
Procedure
1. Take 50ml water sample in a 250 ml conical flask.
2. Add 2 ml buffer solution and 5 drops of EBT indicator.
3. Wine-red colour will appear.
4. Titrate against 0.01M EDTA till the colour changes into bright greenish blue.

Common elements of natural water that significantly contribute to its hardness include calcium and magnesium ions of these elements leached from rocks. Water hardness reduces the utility of water for domestic use. Calcium hardness sometimes may be helpful because it provides a coating in the pipes which protects them against corrosion.
Principle
When calcium and magnesium are present in the water, EDTA reacts with calcium first. EDTA can be used to directly measure calcium when the pH is raised to a point where magnesium hydroxide is mostly precipitated and an indicator that only combines with calcium is employed. At a pH of 12 to 13, several indicators change colour to indicate that all of the Ca has been incorporated by the EDTA. The variations between an aliquot titrated at pH 10 and one titrated at pH 12 or 13 are what define the amount of magnesium.

Principle
Alkalinity of a water sample is can be determined by titrating with standard sulphuric acid. Titration to pH 8.3 or de-colourization of phenolphthalein indicator will indicate complete neutralization of OH and ½ of CO3 while to pH 4.5 or sharp change from yellow to pink of methyl orange indicator, that will indicate total alkalinity (complete neutralization of OH, CO3, HCO3)
Chemicals
1. Phenoplthalein indicator
2. Methyl orange indicator
3. Sodium carbonate, Na2CO3
4. Sodium hydroxide, NaOH
5. Sulphuric acid, H2SO4
Reagents
1. Phenolphthalein indicator: Dissolve 0.5 g in 500 ml 95% ethyl alcohol.
2. Methyl orange indicator: Dissolve 0.5 g of methyl orange in 100 ml of water and dilute to 1000 ml in deionized or distilled water (CO2 free deionized or distilled water should be use).
3. Standard Sulphuric acid, H2SO4 (0.02N): to prepare 0.1N H2SO4 take 3 ml conc. H2SO4 and dilute it in to 1000 ml of deionized or distilled water. Standardise it against standard Na2CO3.
4. Sodium carbonate, Na2CO3 (0.05 N): Take 7g standard Sodium carbonate (Na2CO3) dry at 250 oC for 4 hr and cool in desicator then weigh 5.3 g and transfer it a 1000 ml volumetric flask after that fill to mark with distilled water. Solution should be use within 1 week.

Chemical oxygen demand (COD) is measurement of oxygen demand of waste in terms of the total quantity of oxygen required for oxidation of the waste to carbon dioxide and water. This test is based on the fact that all the organic compounds, with few exceptions, can be oxidized The test is based on the fact that all organic compounds, with a few exceptions, can be oxidized by the action of strong oxidizing agents under acid conditions. Organic matter + Oxidizing agent = CO2 + H2O. The major advantage of COD test is the short time required for evaluation. The determination can be made in about 3 hours rather than the 5-days required for the measurement of BOD
Principle
The oxidizing agents like potassium permanganate and potassium dichromate is usually used to determine COD. Potassium permanganate is selective in the reaction like it reacts with carbonaceous on the other hand it shows nonreactive with nitrogenous matter. Estimating of COD with any method, oxidizing agent must be present to ensure that organic matter is oxidized completely. The organic matter and oxidisable inorganic substances present in water or wastewater get oxidised completely by standard potassium dichromate (K2Cr2O7) in the presence of H2SO4 to produce CO2 + H2O.The excess K2Cr2O7 remaining after the reaction is titrated with ferrous ammonium sulphate [Fe(NH4)2 (SO4)2]. The dichromate consumed gives the 02 required for oxidation of the organic matter.

Free carbon dioxide in the water accumulates due to microbial activity and respiration of organisms. Surface waters normally contain less than 10 mg free CO2 per litre while some groundwater may easily exceed that concentration up to 30 to 50 mg/L.
Principle
Free CO2 reacts with sodium carbonate to form sodium bicarbonate, or with sodium hydroxide to form sodium carbonate. Completion of reaction is indicated by the development of the pink colour characteristics of phenolphthalein indicator at the equivalence pH of 8.3.
Analysis of Water and Effluents
CO2 + N2CO3 + H2O = 2NaHCO3
CO2 + 2 NaOH = N2CO3 + H2O
Reagents
1. Standard sodium hydroxide titrant (0.05N): Dissolve 40g of NaOH in boiled CO2 free distilled water and make up the volume to 1 litre. Filter the solution through a sintered glass filter to remove any Na2CO3, the concentration of solution is 1N NaOH. Store it in in glass bottle.
2. Phenolphthalein indicator

The biochemical oxygen demand is a chemical procedure for determining the amount of dissolved oxygen needed by aerobic organisms in a water body to break the organic materials present in the given water sample at certain temperature over a specific period of time. BOD of water or polluted water is the amount of oxygen required for the biological decomposition of dissolved organic matter to occur under standard condition at a standardized time and temperature. Usually, the time is taken as 5 days and the temperature is 20°C. microbes
C6H12O6 + 6O2 ¾¾¾¾®CO2 + H2O
Principle
The test measures the molecular oxygen utilized during a specified incubation period for the biochemical degradation of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic material such as sulfides and ferrous ion. It also may measure the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand).
Procedure
Fill two BOD bottles with sample (or diluted sample); the bottles should be completely filled.

Dissolved oxygen (DO) refers to oxygen gas that is dissolved in water. Fish are able to absorb oxygen directly from the water into their bloodstream using gills. There are three main sources of oxygen in the aquatic environment: 1) direct diffusion from the atmosphere; 2) wind and wave action; and 3) photosynthesis. Oxygen depletion refers to low levels of DO and may result in fish mortality. A concentration of 5 mg/L DO is recommended for optimum fish health. Sensitivity to low levels of dissolved oxygen is species specific, however, most species of fish are distressed when DO falls to 2-4 mg/L. Mortality usually occurs at concentrations less than 2 mg/L. If fishes gulfing on the water surface and also show the low swimming in the water it indicates deficiency of dissolved oxygen in pond.

The decreased ability of water to transmit light which is caused by suspended particulate matter ranging in size from colloidal to coarse dispersions is refers as turbidity.
Principle
Turbidity is measured by its effect on the transmission of light, which is termed as Turbidimetry or by its effect on the scattering of light, which is termed as Nephelometry. Turbidimeter can be used for sample with moderate turbidity and Nephelometer for samples with low Turbidity. Higher the intensity of scattered light higher the turbidity.
1 mg SiO2/L = 1 unit of turbidity

The total amount of salts dissolved in water is known as salinity. Salinity of water is denoted as ppt, g/L, psu. Salinity of different water has been classified as Freshwater 0-0.5 ppt, Brackish water 0.5-30ppt and Marine water > 300 ppt.
Reagents
1. Silver nitrate (AgNO3) (0.1N): Dissolve 4.25 g silver nitrate in distilled water and make the total volume 250 ml by shaking.
2. Potassium chromate (K2CrO4) indicator (5%): Dissolve 2.5 g potassium chromate in distilled water and make the total volume 50 ml by shaking.
Procedure
1. Salinity in estuarine and coastal water can be estimated by this method.
2. Take 10 ml of water sample in a 50 ml conical flask.
3. Add 3-4 drops of potassium nitrate indicator solution to it.
4. Titrate against 0.1N Silver nitrate till the yellow colour changes to brick red.

Pure water is a poor conductor of electricity. Water may carry electricity fairly well when it contains electrolytes such as Acids, bases and salts. Electrolytes in the solution dissociates with cations and anions and pass on electrical conductivity. Hence, more electrolytes concentration in water, more its electrical conductance. The following ions are crucial for adding conductivity to water.
i) Cations: Mg 2+,Ca2+, Na+ and K+
ii) Anions: Cl-, S4
-, CO3
-, HCO3
-and NO3
-
In the International System of units (SI) the reciprocal of ohm is the Siemens (S) and conductivity is reported as millisiemens per meter (mS/m). 1 mS/m = 10 µS/cm = 10 µmhos/cm and 1 µS/cm = 1 µmhos/cm Principle
The quantity and kind of ions present in a solution affect conductivity, which is a numerical representation of a water sample’s capacity to conduct electricity. Since the majority of dissolved inorganic compounds in water are ionised, they add to conductance. The definition of conductance G is the reciprocal of resistance R.

Pure water is a poor conductor of electricity. Water may carry electricity fairly well when it contains electrolytes such as Acids, bases and salts. Electrolytes in the solution dissociates with cations and anions and pass on electrical conductivity. Hence, more electrolytes concentration in water, more its electrical conductance. The following ions are crucial for adding conductivity to water.
i) Cations: Mg 2+,Ca2+, Na+ and K+
ii) Anions: Cl-, S4
-, CO3
-, HCO3
-and NO3
-
In the International System of units (SI) the reciprocal of ohm is the Siemens (S) and conductivity is reported as millisiemens per meter (mS/m). 1 mS/m = 10 µS/cm = 10 µmhos/cm and 1 µS/cm = 1 µmhos/cm Principle
The quantity and kind of ions present in a solution affect conductivity, which is a numerical representation of a water sample’s capacity to conduct electricity. Since the majority of dissolved inorganic compounds in water are ionised, they add to conductance. The definition of conductance G is the reciprocal of resistance R.

Principle
The estimation of all solids, including dissolved, volatile, and suspended solids, is called total solids (TS). The residue that remains after the unfiltered sample evaporates between 103 and 105°C can be used to estimate total solids. Total dissolved solids (TDS) and Total suspended solids (TSS) are its two constituent components. After heating to 550°C, each fraction is separated once more into fixed solids and volatile suspended solids (VSS). The percentage of ash that remains is called fixed solids, while VSS is the organic proportion that was lost as CO2.
Apparatus
1. Ceramic or platinum evaporating dishes 100 mL volume.
2. Dessicator contain CuSO4.5H2O. Desiccator, provided with a desiccant containing a colour indicator of moisture (CuSO4.5H2O).
3. Analytical balance
4. Hot air oven
Procedure
1. Take a clean evaporating beaker or dish of required volume and dry it at hot air oven at 103 to 105oC for 1h, then cool and store it in a desiccator until needed. Immediately weigh before use. Note » the initial weight (Wi) in mg.
2. Take 250-300 ml or required volume of unfiltered well mixed sample in it (beaker or dish).
3. Place it in hot air oven at 103 to 105 oC for 2 h.
4. Cool it in a desiccator and take the final weight (Wf) in mg.
5. Replication the above process until a constant weight is achieved