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biology lab manual class 11- experiments
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  120  Aim:  To detect the presence of albumin in the given sample of urine. Principle:  Nitric acid causes the precipitation of albumin. When heated or treated withsulphosalicylic acid, albumin undergoes coagulation. Requirement: Glasswares: Test tubes, graduated pipette (5 mL capacity), spirit lamp;Chemicals: Concentrated nitric acid, acetic acid, Robert's solution, sulphosalicylic acid or asolution containing 13% salicylic acid and 20% sulphuric acid; Miscellaneous: test tube stand,test tube holder. Exercise 31 Procedure  (a)Nitric acid ring test Take 5 mL of concentrated nitric acid in a test tube.ãIncline the tube and add the urine sample with a dropper, sothat the latter flows down slowly along the side of the test tubeto form a separate layer.ãA white ring develops at the junction of the two liquids whichindicates the presence of albumin in the urine sample.  OR ãTake about 5 mL of Robert's solution in a test tube.ãNow incline the test tube and add 2 to 3 mL of the given sampleof urine by means of a dropper along the inner side of the test tube so that it forms a layer over the Robert's solution.ãThe presence of white ring at the junction of two layers indicatesthe presence of albumin in the sample. (b)Heat coagulation test ãTake about 6 to 8 mL of urine in a test tube.ãIncline the test tube at an angle and heat the upper one-thirdof the test tube by a low flame.ãTurbidity develops in the heated portion of the urine.ãAdd 1% acetic acid drop by drop and boil or simply add a drop of 33% acetic acid.ãIf the turbidity persists it confirms the presence of albumin inthe urine sample (disappearance of turbidity, confirms thepresence of phosphates).  121 (c)Sulphosalicylic acid test ãTake 3 mL of urine in a test tube.ãAdd a few drops of sulphosalicylic acid and heat it gently.ãA whitish or cloudy turbid solution or precipitate (coagulation)in the solution indicates the presence of albumin in the urinesample. Discussion  A trace of protein which is less than 250 mg (in 24 hours urine) is found innormal urine. Under pathological conditions like albuminuria, albumin isfound in urine above normal level. This amount is so negligible that it escapesdetection by any of the simple test. In kidney disturbance and in high bloodpressure, albumin level in urine is significantly high. Questions 1.What is the colour of the urine and name the pigments responsible for thischaracteristic colour?2.In which organ of our body, highly toxic ammonia is converted into urea?3.Name the disorder that shows presence of excess urea in the urine.4.Name the disorder in which glucose level is high in urine.5.What do you call those animals that eliminate nitrogen mainly in the form of urea?6.Which other organ of our body also excretes urea in small amount?7.Which reagents will demonstrate presence of protein in urine?8.What is the significance of appearance of different colours while performing Benedict'stest?9.What is the significance of performing Seliwanoff's test? Exercise 31  122  Aim:  To detect the presence of bile salts in the given sample of urine Principle:  Old and damaged RBCs are removed from the circulation mostly in the spleenand to some extent in the liver by macrophages. Hemoglobin of the RBCs is broken down inthe cytoplasm of macrophages. When iron is removed from heme component of hemoglobin,the iron-free portion of heme is converted to biliverdin, a green pigment, and then intobilirubin, a yellow orange pigment. Bilirubin enters the blood and is transported to the liverfrom spleen. In the liver, bilirubin is secreted by liver cells into bile, which passes into thesmall intestine and then into the large intestine. Bilirubin is detected in urine in certainpathological conditions only. Requirement:  Test tubes, measuring cylinders (10 ml), funnel, dropping pipette or drop bottle,Lugol's iodine solution, barium chloride solution (10%,) Fouchet reagent, sulphur powder,concentrated nitric acid, test tube holder, test tube stand, cotton, filter paper, distilled water. Exercise 32 Preparation of reagents (i)Lugol's iodine solution: Dissolve 1g of iodine crystals and 2 g of potassium iodide in 100 mL of distilled water.(ii)Fouchet reagent: Dissolve 25 mL of trichloroacetic acid (TCA) in75 mL of distilled water. Now add 1g of ferric chloride to this solutionand mix.(iii)10% Barium chloride solution: Dissolve 10 g of BaCl 2  in 90 ml of distilled water and make up the solution to 100 mL with water. Procedure (a)Lugol's Iodine Test Pour 4 ml of urine sample into a test tube. Add 4 drops of lugol'siodine solution to this tube. Shake the tube well and observe. A faint yellow to brown colour indicates absence of bile pigments while light to dark green colour indicates the presence of  bile pigments. (b)Gmelins Test  Take 5 ml of concentrated nitric acid in a test tube. Add an equal volume of the given urine sample to it slowly along the sides of   123 the test tube. Formation of a green, blue, yellow or red ring at the junction of the two solutions indicates the presence of bilepigments. Procedure Bile pigments present in the urine react with concentrated nitric acid andinduces formation of a coloured ring at the junction between the urine andacid layer. (c)Fouchets Test  Take 5 mL of the given urine sample. Add 2-5 mL of BaCl 2  to this test tube and mix the two solution. A precipitate will appear. Now filter the mixture. The precipitate containing the bile pigments remains onthe filter paper. Add 2 drops of Fouchet reagent to the precipitate onthe filter paper. If the precipitate turns green, it shows the presence of  bile pigments. Discussion  The colourless bilirubin is oxidised by the ferric ion of ferric chloride (present in the Fouchet Reagent) to green biliverdin.Bilirubin + Fe +++ Biliverdin + Fe ++ (colourless)(green) (  Exercise 32 Questions 1.Give the names of the pigments found in bile.2.Which organ of the body produces bile pigments?3.Which pigment provides colouration to the bile?4.What are the functions of bile pigments?5.How are the bile pigments produced?6.Mention the name of the diseases during which excretion of bile pigments occurs inurine.7.What are the different tests to detect the presence of bile pigments in urine?
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