TRAINING REPORT TOPIC: Quality Assurance & Microbiological testing Submitted by: Saumya Singh Course- M.sc Microbiology Meerut institute of Engineering & Technology , Meerut Parag Dairy Submitted to: Mr. ~ Sudhir Rathi Head of Quality Assurance in DUGDH UTPADAK SAHKARI SANGH Ltd, Gangol road Partapur , Meerut- 252103Certificate ; icrobiological This is to certify that the report entitled ‘Quality Control ee ecah : f the work done by Ms. Sau! Testing of Dairy Products’ is a summary of : under the Training Program at the Quality Control Laboratory at Dairy during the period of 2 months i. e. 24-08-2022 to 24-10-2022. (Coordinator) : Mr. Sudhir Rathi In-charge Quality: Mr. Sudhir Rathi Acknowledgement First of all ,| would pay regards to the almighty who has led me to the patch of science and inspired to explore opportunities that come in the offering. All this would not have been Possible without the consent, support, encouragement and blessings of my beloved parents it's my privilege to express my gratitude to miss Vandana Singh manager ( administration and HR Gangol Sahkari Dugdh Utpadak limited plant C/o gangol road partapur, meerut uP.) for glving me permission to undertake the dissertation work in the prestigious company in the partial fulfillment of the degree of master science in microbiology.| also over gratitude to Mr. Sudhir Rathi head of the qual staff of dairy plant for their advice, support, disarming, of my dissertation. | heartily extend my gratitude to Dr. Shalini lity assurance department and all QC frankness and support in completion Sharma , former Principal of department of Microbiology and Biotechnology MIET, Meerut for always encouraging to study and giving their valuable advice towards my career. A spec CONTENT TOPICS * Company Profile + Milk Products of Parag * Compositions + Quality Control * Quality assurance * Milk adulterants * Product Testing * Microbial Analysis of Milk * Discussion Conclusion References cial thanks to my family and friends as well. Saumya SinghCompany Profile | About the Company The common brand name of the company is “PARAG” the meaning of PARAG is the pollen of flower the slogan in the logo is: - PURE NATURAL & GOOD HEALTH parag milk shed is situated in the Lucknow, the capital of Uttar Pradesh since independence ithas formed part of the traditional supply ine of agriculture products from the village to the big cities rich in its milk potential the milk shed has, in the source of last few decades ploited by small traders and powerful contractors and well organized while such intermediaries were retaining large profits the rural milk day by day. In 1950-a co-operative milk supply village and supplied to been thoroughly ex private dairies. Thus, producers found their position deteriorating union was organized in Lucknow , which started collecting milk from arkets, This milk union continued function for about a decade, in the Lucknow and local mi dia in1959-60 to mean time Lucknow milk scheme was established by government of In ensure cheaper milk to the local pollution of Lucknow. The scheme started operating through 12 chilling centers in Eastern Uttar Pradesh, These chilling centers were mainly coated in thither district of Lucknow , Barabanki, Raebareli , Kanpur, Unnao, Sitapur, Meerut etc . The milk was mainly collected through contractors. 10 milk unions were also found ‘almost at the same time ,around each chilling center . These continued functioning in a rather lop-sided manner till 1977.Gradually all the milk union almost become defunctioning and was supplying penis quantity of milk during the years 1970-77. Obviously contractors had monepely a tollected major share of milk which was either supplied to Lucknow or to the local ys population of the city. This programmer was launched in Uttar Pradesh in 1972 and the implementing agency in the was pradeshak cooperative dairy federation limited. ‘The work of expanded dairy started functioning on full capacity in 1991-1993 year: The liquid milk and products are selling in the market in the brand name of PARAG. The milk product has been marketed by D.US.S Meerut. The sale of liquid milk has been carried out Meerut Producer’s-operative Milk Union Ltd, Meerut. Mostly unit milk Sahakari Board where connected under Operation Flood ~ Il, having the name Dugdh Utpadak Sahakari Sangh (D.USS.) Ltd. P.C.D.F. Ltd. takes royalty of common brand name PARAG and all the important policy taken b y Pradeshik Cooperative Dairy Federation Ltd. Who monitors to all the 0.U.5.S Ltd. i.e, Meerut , Lucknow ,Kanpur, Varanasi. PARAG provides hygienic, nutritious milk and milk product. In the year 19830peration Flood - II scheme was launched, the main objectives of the Operation Flood were following -- To collect the milk directly from the producers (Villagers through society). To insure the supply of quality milk collected from the villagers which being sold in the market area of city.- To save the producers, villagers and the customers from the middle man.: The milk is collected firstly to the society level then it comes to D.U.S.S. level finely it comes under the state level i.e. Federation Objectives of the Company OBJECTIVES PCDF’S front-end objective was to see that there was enough milk for everyone in town. Marketing is simply the PCDF’S tool to achieve their ultimate objective and delivering the pure Parag milk to every home. PURPOSE pas ue to build a system to ensure that individual farmer got a fair price for the milk they sold. Management System Policy ae firmly believe that the path to progress can only be through procurement and production of superior quality milk & milk products, timely deliveries & total customer satisfaction. We oD bring paaiao change in the lives of our milk producers by ensuring profitable returns to their produce and offering quality i aul 8 quality & safe products at competitive price tothrough proffesional excellence. We will create a vibrant work environment, continuously i mroving uponour production, marketing and service operations through application of quality, food safety &environmental management systems. Fundamental Knowledge of Milk Milk is the secretion derived after complete milking of healthy milk animal, excluding that obtained within 15 days before or after 10 days of calving. Composition of Milk 01. Milk Fat Milk fat is the mixtures of 19 fatty acids. The bulk of fat in milk exist in the form of small globules with size approx. 0.1 to 22microns. It is an oil-in-water type emulsion. 02.Proteins Protein is the most complex organic substances. They are vital for living organisms as they constitute an indispensable part of the individual body cell. The protein of milk consists of Casein, Lacto globulin and Lacto albumin. It is in colloidal state. Casein is only found in milk. It is easily coagulated by heat treatment. 03.Water It provides the medium in which all the milk constitutes are either dissolved or suspended. Most of it is free and only a small portion is in bound form, being firmly by protein and phospholipids. 04.Lactose(Milk Sugar) It is found only in milk, It exist in true solution phase and is fermented by bacterial to yield lactic acid. It is very important for cultured milk products. It can also cause souring in milk and its products.05.Mineral Matter Although its present in small quantity it is very essential for human body. ue as mostly salts like Mg, Na, P, N ete. It influences physio-chemical properties of milk and also affects the nutritive value. 06.Phospholipids: There are three types of phospholipids (Lecithin, Cephalic and Sphingomyelin). Lecithin is important constituent as it helps in the formation of outer membrane of fat globules.07.Pigment: There are two type of pigment present in milk, Fat Soluble and Fat insoluble. Carotene is fat soluble and is responsible for the yellow colour of milk. The other two are Xanthophylls and riboflavin. Carotene also acts as an anti oxidant and as a source of Vitamin A. 08.Milk Enzymes The important milk enzymes and their specific action are as follows: + Anise (desaturase)-Starch +, Lipase Fat splitting, leads to rancid flavour * Phosphatase It is capable of splitting certain phosphoric esters, * Protease It is capable of splitting proteins. — * Catalase decomposed hydrogen peroxides. 09.Vitamins Vitamins are present in minute quantities in milk or any other food but play a very important role in vital functioning of human body. There are 25 vitamins present in milk. These are fat soluble e.g. Vit. A, D, E, K, apart from fat soluble there are water soluble vitamins as B- complex(B1, riboflavin or 82, pantothenic acid, niacin, pyridoxine or B6.)Milk Products of Parag + Packaged Milk Pasteurised and standardised packaged milk is available types, viz., Parag Gold (full cream milk), Parag Taaza (tone Double Toned Milk. Flavoured Milk is also available in 200 ml packs.2. in 500 ml and 200m! packs of four .d milk), Paragliter (skimmed milk), + Butter It contains less than 80% milk fat and more than 15% moisture and high acidity. It is prepared exclusively from milk cream of curd of cow or buffalo milk without the addition of salt, colour or any preservative and is intended for cooking or for preparation of Ghee. Parag Butter is available in 20 \gm, 100 gm and 500 gm packs.3. * Ghee: ‘About 43% of total quantity of milk produced in India is manufactured first into butter and then converted into Ghee. Bulk of Ghee is derived from buffalo milk because itis richer in muted in steam jacket kettles. Which are fat that cow milk. In Parag surplus butte equipped with mechanical stirrers and heated with steam till the moisture is removed. * Dahi : Commonly known as Yoghurt. Two type of dahi is available viz., curd and sweetened curd (Mishti Dohi) in 200 gm packs.5. + Paneer: Commonly called as Cottage Cheese. In Parag, Paneer is produced by thetraditional method in which citric acid is added to the boiled milk and the milk immediatelygets adulterated and water is separated and paneer is obtained. It contains less than 50% fratof more than 60% moisture.6. * Others: Other Parag products avaialable in the market are: Peda, Cream, Ice-cream, Kheer (chhena and rice), Rasogulla, Gulab Jamun, Besan laddu.\ESSENTIAL COMPOSITION IN MILK BUFFALO COMPONENT COW MILK (gm) nen FAT 46 6.6 | MOISTURE 18.4% 22% PROTEIN 3.4 3.9 WATER 86.6 84.2 LACTOSE 49 49 PH 6.5 6.7 ACIDITY 0.13 0.75 ESSENTIAL COMPONENTS IN GHEE COMPONENT COW MILK | BUFFALO MILK| GHEE GHEE FAT % 99-99.5 99-99.5 MOISTURE (%) <0.5 <0.5 NUTRIENT COMPOSITION IN DAIRY MILK[mK TyPE| FAT(%) | SNF(%) | ACIDITY (%) FCM 6.0 9.0 0.13-0.14 STD 4s 8.5 0.13-0.14 | DTM 15 9 0.13-0.14 PTM 3 8.5 0.13-0.14 AVERAGE COMPOSITION IN BUTTER PROTIEN) FAT | CARBOHYDRATES VITAMINS] MINERALS} WATER 1% 82% 0.5% 05% A,D| 2% 14% SODIUM. AVERAGE COMPOSITION IN CREAM(%) : “Components Half-half cream | Table cream Whipping cream MOISTURE 80.3 73.8 57.7 | PROTEIN 3.0 27 2.0 FAT 11.5 19.3 37.0 CARBOHYDRATES 43 3.6 28 ASH 07 06 05 Quality control is an essential and most important dey every organization has efficient quality control syste! uality Control partment for any manufacturer. Today m. Quality control is depend upon onlyt Procedure). In D.U.S. Ltd. LUCKNOW at reception point is tare of milk from different societies (Producers Villagers) Milk is collected and Eee A carried out quickly after cleaning it is send for further processing. Finally 2 a posteurs ae three type of milk obtain that is Full cream milk. Toned Milk, Janta Milk .Milk pr: through some stages— practical{Survey Analysis and Right 1.Organo Leptic Test It passes through three stages. This is the first stage :Seeing Smelling Testing 2. MBRT (Methyl Blue Reduction test)it is done to check the hygenic quality of milk. It gives an idea about the bacterial load inmilk.10 ml milk is taken in a test tube along with 1 ml of Methyl blue. It is kept for observation at37 deg. Celsius. Bacterial load is inversely proportional to disappearnce of blue color. 3. Gerber Method It is done for fat content of milk 10 ml mik is taken in milk butyrometer. 10 ml conc. H2S04(1.0-1.182 specific gravity) is added to it. 1 ml Amyl alcohol (0.85 specific gravity) is added. Lock stopper is put on butyrometer and it is centrifuged for 3-5 mins. Scale reading is observed 4, SNF(Solid not Fat) Analysis SNF=CLR(Corrected Lactometer Reading)/4 + 0.2*Fat + 0.29 (This formula is used for Big lactometer) SNF=CLR/4 + 0.2*Fat + 0.50 (for small lactometer) 5. Acidity test This is done to find out the pH of milk. Acidity of milk should be between 0.10 = 0.15. Curdling occurs at 0.18. 9ml milk is taken in a beaker. 1% Phenophtahlein indiactor is added. 0.1N NaOH is take to be titrated against. Acidity=9*N*V(vol of NaOH)/w(milk). UALITY ASSURANCE Quality assurance (QA) is any systematic process of determining whether a product or service meets specified requirements. QA estab shes and maintains set requirements for developing or Manufacturing reliable oducts. Products. A quality assurance system is meant to increase customer confidence and aic it enables a ,, while also improving work processes and efficiency, and it company's credi company to better compete with others. behind QA The ISO (International Organization for Standardization) is a driving force bel i i ith the practices and mapping the processes used to implement QA. QA is often paired wi se ISO 9000 to ensure that their quality Iso 9000 international standard. Many companies u assurance system is in place and effective. The concept of QA as a formalized practice started in the manufacturing industry, and it has since spread to most industries, including software development. Importance of quality assurance Quality assurance helps a company create products and services that meet the needs, expectations and requirements of customers. It yields high-quality product offerings that build trust and loyalty with customers. The standards and procedures defined by a quality assurance program help prevent product defects before they arise. Milk Adulterants * Soda 2 mlof milkis taken in a test tube.2ml rosalic acid solution (0.05N) is added t it.f red color appears ~ soda +ve If pale ornage color appears — soda -ve * Urea 2 mi milkis taken in a test tube.2 ml DMAB (dimethyl benzaldehyde) solution is added it If light color (yellow) ~ urea -velf dark color (yellow) — urea 4ve eee * Salt1 ml of milk is taken in a test tube.5 drops of salt detecting sol.(Potassium chromate)added5 mil AgNO3(0.1342%) is added .Yellow color ~ salt +ve Brown color ~ salt -ve + Glucose 41 mi milk is taken in a test tube.1 ml glucose detection sol. A (Barford sol) added Gave boiling water bath for 3 mins immediately dipped in cold after 3 mins Added 1 ml Solution 8 (Phosphomonolytic acid) dark blue color ~ glucose +ve Light blue color — glucose -ve Precaution-In this test the test tube should not be subjected to heat for more than 3 minutes while giving water bath .This can result in a false positive result. Sugar 1 mi milk is taken in a test tube2ml sugar detecting sol.(resorcinol) is added Kept in water bath for 1 — 3 mins Brick red color — sugar +ve Light color ~ sugar -ve Starch 3m milk is taken in a test tube It is boiled and then cooled4-5 drops of starch detecting sol(iodine sol.)added Yellow color ~ starch -ve Blue color ~ starch +ve ‘Ammonium Compound ‘Sml milk is takeniml . Comp. Detection sol .Light shade ~ test -ve Dark shade ~ test +ve. Product Testing ? Curd Acidity of Curd ‘Sgm curd is weighed.10 ml distilled water is added It is titrated against 0.1 N NaOH .As per experiment, 9.7 ml NaOh was consumed, hence acidity of curd was 0.97. Butter Moisture in Butter: Empty Beaker was weighed: 51.7036 gm10 gm butter was taken Moisture was boiled off. Weight if butter = 59.743-51.7036= 13.57 %Standard moisture should not be more than 16%. Acidity of butter Butter was melted and 040 gm Implies, mixture % =:20 gm butter is weighed.90 ml d. w. Is added to it. Butter is melted = 9°0,02"1.5/20 = 0.01 Standard acidity should not be more than 0.05 Acidity = 9*N*V/w Salt in Butter: Vapor is boiled off from 10 gm butter. Petroleum ethe! The seperated fat is weighed: 16.8693 gm.250 ml distil in water.17.6ml of solution is taken in a seperate beaker. 5 d added. 0.04 mlof AgNO3 is added. Standard salt content shoul 1 is added due to which fat seperates. iled water is added. The salt dissolves ops of K2Cr207 (0.1204 N) is Jd not be more than 2-2.5%. Paneer Acidity of paneer: | phenophthalein is added to it 9.gm paneer is taken and distilled water is added to it.1 ml consumed, hence acidity is 0.13. and it is then titrated against 0.1 N NaQH.1.3 ml NaOH is ci Ghee 53,9982 gm Weight of beaker with ghee: 69.643 gm Weight if beaker 169 gm Implies, Moisture content is Beaker is weighed: with paneer: 74.6433 gm Weight after boiling off moisture: 71.664 74,643371.6699=2.9764 moisture is present in 5 gm paneer .Implies, 59.52%, thus in 100 gm, 40.480 gm is dry weight. © Fat test: Same as Gerber method of fat analysis. Microbiological Analysis of Milk Purpose: To test the liquid milk-Standard Plate Count (SPC) Equipment used: 1) Laminar Air Flow 2) Sampling Bottle 3) Auto pipette with tips 4) Dilution Blank (9 ml or 99 ml) 5) Test Tube/Culture Tube 6) Plating Media 7) Durham's Tubes 8) Petri Dish9) Hot Air Oven 10) Autoclave 11) Incubator 12) Colony Counter 13) pH Strips 14) Refrigerator Sterilization: Sterilize sampling and plating equipments whenever possible with dry heat in a hot air oven at 170°C for two hours. Sterilize the media and materials that are likely to be charred in dry ‘oven, by autoclaving at 15 psi (127°C) for 20 minutes. After sterilization all media should be stored in hygienic condition. Collection of sample: Milk sample received should be stored in refrigerator below 7°C till tested. General Precautions: a) Use only sterilized bottle to collect sample b) Collect all samples aseptically. c) Stoppers or lid of the bottles should not be removed from the bottles unless necessary. 4) Replace the stopper or lid immediately after the sample is obtained. e) Do not fill the bottle more then three fourth of its capacity. f) Clearly label each sample bottle indicating the source, date etc. g) Keep the sample immediately in refrigerator below 7°C. Composition of Medi Plate Count Agar (Tryptone Glucose Yeast Agar) For S.P.C. Ingredients: 1) Tryptone 5.0 gm / litre2) Glucose (Dextrose) 1.0 gm. /litre 3) Yeast Extract 2.5 gm/ litre 4) Agar 15.0 gm. / litre 5) Final pH 7.0 + (0.1) Preparation: Suspend 23.5 gm of ready made Plate Count Agar in 1000 mi distilled water, add 6.5gm NaCl, dissolve and adjust the pH 7. Boil to dissolve the medium completely and fill it in flask or bottles. Sterilization: Sterilize by autoclaving at 15 Ibs pressure for 20 minutes. At the time of usecompletely melt the media, then cool it in water bath set at 45°C. STANDARD PLATE COUNT METHOD Materials required: 1) Plating chamber 2) Petri plates 3) Plate count agar 4) Auto pipette with tips (1.0)/pipette 2.2 mI.5) Dilution tubes containing 9 ml of batch phosphate buffer solution. Procedure: 1-Transfer 1 ml of well mixed sample to 9 mi diluents (phosphate buffer solution). MixWell and transfer 1 ml of this suspension to second tube to make second dilution, Similarly make third and fourth dilution as per requirement 2-Arrange the Petri plates for each dilution to be tested, mark them with sample no. and Date. 3 Transfer 1 ml in each plate from respective dilution of sample being tested . 4-Melt the plate count agar, cool it about 4: 5°C and pour 10 ml of this medium into the Petri dishes. Mix the agar by rotating the plate,s.Allow the agar to set, invert and incubate the plates at 37°C for 48 hours g-at the end of 48 hours remove the plate from the incubator and count the colonies 7-Also prepare a control plate with 15 ml media for checking its sterility Counting and Expression Of results: Count the colonies grown in Petri plates. Count only those plates, which have 30 300colonies. Computation: Count the colonies developed in each plate for respective dilution. Multiply colonies per plate by dilution used and report the arithmetic average as plate count per milk Recording / Reporting Of Result /Record and report the result as — Standard Plate Count/ml/gm---Or SPC/ml/gm EFFLUENT WATER-TREATMENT Water is used for chilling the milk so that bacterial action in milk can be reduced. There are many impurities present in the water. There is several treatment of water for reduce these impurities &turbidity from the water. EFFLUENT TREATMENT PLANT (ET P) Dairy effluent are organic in nature which have high BOD when it is discharge as such in water bodies the bacteriological action will deplete the oxygen content of stream which is not good for animals ,fishes & plants. Life which depend on oxygen . Therefore, it is necessary to remove the organic matter content prevent in the effluent before discharging it into the environment .The ETP have following stages:-1: Oil collection sump/ fat removed unit:- It is a devices which is used to remove out fat, oil, other greasy substances and floating materials from the effluent water coming out from the dairy industries.2:Equalization tank /Neutralisation tank :-Diffused aeration line provided in the equalization tank. Diffused aeration prevention the formation of lactic acid and reduced BOD about 50%. Neutralization tank are meant for to control the pH of effluent. Due to this, the acidity of effluent water is reduced and it becomes harmless for the next stage treatment.3:-Aeration Tank:-In this tank extended are provided. n this tank, with the help of micro-organism,, in the presence of air the organic matter is converted into simpler material through Inoculation Process and water becomes less harmful. In this tank activated sludge takes place. The Sludge digestion takes through aerobatic process. The sludge new active microbial cell here which are common by referred as mixed liquour suspended solids which is golden brown in ‘clour, and a part of the sludge with the old cell in sent for clarifier /sludge setting tank.4:. 1000 ml). Keep ina brown glass stoppered bottle. B Alkaline Potassium iodide solution Dissolve 100g of KOH and 50g of KI in 200ml of boiled distilled water. ¢ Manganous sulphate solution Dissolve 100g of MnSO4.4H20 in 200m! of boiled distilled water and filter. D.Starch solution Dissolve 1g of starch in 100m! of warm(80'c-90'c) distilled water and add a few drops of formaldehyde solution. E.sulphatic acid H2S04.conc(sp. Gr.1.84 ) PROCEDURE: LFillthe sample in a glass stoppered bottle (BOD bottle) of known volume (100-300) ;arefully, avoiding any kind of bubbling and trapping of the air bubbles in the bottle after Placing the stopper. pour ml of each MnSO4 and alkaline KI solution (in case, the volume of the sample is about 300ml, instead of 1ml of reagents add 2m! solution of each),well below the surface from the walls, The reagents can also be poured at the bottom of the bottle with the help of Special pipette syringes to ensure better mixing of the reagents with the sample. Use always, Separate pipettes for these two reagents. A ppt. will appear 3Place the stopper and shake the well by inverting the bottle repeatedly. Keep the bottle for some time By settle down the ppt. If the titration is to be prolonged for few days, keep ‘he sample at this stage with the ppt. “Add 1-2ml of cone. H2S04 and shake well to dissolve the ppt. part of them (50-100m!) in conical flask for SRem, of love ontents, oF 2 ve either the whole cont! mixing of O*yBe tit ; “ation, Prevent any bubbling to avoid fusie the content, within one hour of g f ion usi iss ‘sl hate solution using starch as an indicator an Of the ppt. against sodium eee colourless “At the end point, initial dark blue colour pevUaTION: When whole contents have been titrated: Diss, 0 . xygen, (Mg/I= (mI*N) of titrated*8*1000/Vi-v apenarly a part of the contents has been titrated: Diss. Oiygen, Mg/l= 9mI*N) of titrant*8*1000/V2(V1-v/V1) where, yievolume of sample bottle after placing the stopper. \nevolume of the part of the contents titrate dv-volume of MnSO4 and KI added In ‘occanography , the unit ml/I is preferred over mg/l. It can be obtained by dividing the value inme/l. | AIDE MODIFICATION OF WINKLER METHOD in oxidizing and reducing material may effective interface with the determination of oxygen by converting iodide ions to iodide or vice-versa. The aide modification removes the interference of such substances especially nitrite. Nitrite is destroyed by sodium azide . The method, therefore is suitable particularly in polluted waters, biologically treated waters and in BOD samples. | PRINCIPLE:- The presence of certa REAGENTS A Sodium thiosulphate ,0.025NSee winkler’s method 8 Alkaline iodide amide solution ed water to make 1 It. of solution. KOH and 1508 of distill Dissolve 500g of NaOH or 7008 of water, Mix the two solutions (a)&(b). ))Dissolve 10g of NaN2 in 40m! of distilled thod © Manganous sulphate solution See winkler,s met 2 Starch solution See winkler’s method € Sulphate aciahi2s04. cone. (sP- 6°84)wv cedure: Follow the same Winkler’s method except that , add alkaline iodide azide Maton instead of alkaline potassium iodide in the same quantities. cALCULATIONS= Same as for Winkler’s iodide method. BIOCHEMICAL OXYGEN DEMAND(BOD)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 the stabilizing the biologically degradable organic matter under aerobatic conditions The principle of the methods involves, measuring the difference of the oxygen concentrated between the sample and after incubating it for Sdays at 20’cAPPARATUS & REAGENTS A.BOD bottles B.80D incubator Having temp. control at 20'cC.Phosphate buffer Dissolve each 82.5g KH2PO4. 21.75g K2HPO4. 33.4g Na2HPO4.7H20 and 1.7 g NHACLin distilled water to prepare 1 It. of solution, Adjust pH to7.2.D.Magnesium sulphate Dissolve §2.5g MgS04.7H20 in distilled water to prepare 1 It. of solution. E .Calcium chloride Dissolved 27.5¢ of anhydrous CaCI2 in distilled water to prepare 1It. of solution. F.Ferric chloride Dissolve 0.25 g FeCl2.6H20 in distilled water to prepare 1It. of solution. G.Sodium sulphite solution, 0.025 NDissolve1.575g Na2SO4 and dilute to 100ml. Solution should be prepared freshly. PROCEDURE:- 1:- Prepare dilution water in a glass container by bubbling compressed air in distilled water for about 30 minutes. 2: Add Iml each of phosphate buffer, magnesium sulphate, calcium chloride, and ferric chloride solutions for each It. of dilution water and mix thoroughly. 3s Neutralize the sample to pH around 7.0 by using 1N NaOH or H2S04. ‘4z-Since the DO in the sample is likely to be exhausted ; itis usually necessary to prepare a suitable dilution of the sample acc. to the expected BOD range. See the table for the dilution of the sample. SPrepare dilution in a bucket or a large glass trough , mix the contents thoroughly. fill 2 sets of BOD bottles. 6: Keep one set of the bottles in BOD incubator at 20’ for § days, and determine the DO contents in another.