Biochemistry Lab Report PDF

CARBOHYDRATES 1.1 Isolation of Liver Glycogen = white precipitate 5% TCA - Used to rinse sand and mortar and pestle 10% TCA - Used with washed sand to grind liver tissue - Used as a precipitating agent 95% ethanol - Added to the tissue liver extract in a ratio of 2:1 1.2 Isolati...

CARBOHYDRATES 1.1 Isolation of Liver Glycogen = white precipitate 5% TCA - Used to rinse sand and mortar and pestle 10% TCA - Used with washed sand to grind liver tissue - Used as a precipitating agent 95% ethanol - Added to the tissue liver extract in a ratio of 2:1 1.2 Isolation of Plant Starch Plant source is cut and grinded with 1mL water. Add 30mL water and mix well. Strained using cheesecloth Discard residue and collect FILTRATE Decant solution slowly and wash once with water using the DECANTATION process. Appearance of precipitate depends on the plant source used. 1.3 General Tests for Carbohydrates 1.3.1 Molisch Test 1-2 mL of 1% carbohydrate solution + 2-3 drops of Molisch reagent. Mix contents. Incline tube and carefully pour 1-2mL conc. Sulfuric acid. Note the formation of layers Principle: Carbohydrate undergoes dehydration upon introduction of conc. H2SO4 to form furfural. Furfurals then condense with sulfonated alpha-naphthol to give a purple or violet coloured ring at the junction. Is also a general test for the presence of carbohydrates and the presence of any monosaccharides gives a positive result. Molisch reagent is a mixture of a-naphthol and ethanol. 1.3.2 Anthrone Test 1mL of 1% solution + 2mL of Anthrone’s reagent. Heat and note the change. Cool and record the result. Principle: Same principle with Molisch Test except that the furfurals and hydroxy-methyl furfurals give condensation products with anthrone that are bluish-green in color. 1.3.3 Iodine Test 1mL of 1% solution + a few drops of 0.01M iodine solution. Heat and note change.  Cool and record result. Principle: Used to test the presence of starch. Starch turns into bluish-violet color upon the addition of aqueous solutions of the triiodide anion, due to the formation of an intermolecular charge-transfer complex. Absence of starch = brown color of the aqueous sol’n. Reagent used in the iodine test is Lugol’s Iodine. 1.4 Specific Reaction Tests for Carbohydrates 1.4.1.1 Seliwanoff’s Test 3 test tubes -> 1mL of each sugar solutions(glucose, xylose,fructose) + 5mL Seliwanoff’s reagent each tube. Immerse in a boiling bath. Observe color changes during the first 15mins. of boiling. Record color and time of formation for each solution tested. Principle: Seliwanoff’s test is a chemical test which distinguishes between aldose and ketose sugars. When heated, ketoses are more rapidly dehydrated than aldoses. Ketose = Fructose -> deep cherry red color. Aldoses = Glucose, Xylose -> faint pink to cherry red color if the test is prolonged. 1.4.1.2 Bial’s Orcinol Test 0.5mL of each sugar solution -> separate test tube + 4.5mL Orcinol reagent. Place tubes in a boiling water bath. Note the color formed with each reactant during 15mins. of heating. Principle: Under hydrolysis pentosans are hydrolyzed into pentoses. Pentoses are dehydrated to yield furfural, which in turn condense with orcinol to form blue-green precipitate. The intensity of the precipitation is directly proportional to the concentration of the pentoses in the sample. The intensity of the color developed depends on the concentration of HCl, ferric chloride, orcinol and the duration of boiling. 1.4.2.1 Mucic Acid Test 50mg of sugar in 4 test tubes + 1mL distilled water + 1mL conc. nitric acid to each tube. Heat tubes in boiling water bath for 1 hour. Add 5mL distilled water and let tubes stand and cool slowly. Induce crystallization by scratching the tube with a clean stirring rod. If no crystals appear, let it stand until the next lab period. Examine and draw the crystals under the microscope. Principle: Highly specific and is used to detect the presence of galactose and lactose. Monosaccharides upon treating with potent oxidizing agents like nitric acid yield saccharic acids. Nitric acid - capacity to oxidize both aldehyde and primary alcoholic groups. 1.4.2.2 Osazone Formation 5mL sugar solution -> test tubes 4g sodium acetate + 2g phenylhydrazine HCl + 3-10 drops glacial acetic acid. Warm with stirring until solution clears. Add 2mL hot solution to each sugar solutions. Stopper with cotton, mix well and heat in a boiling water bath for 30mins. Cool at room temperature and examine crystals under the microscope. Note the time that crystals develop in each solution. Draw the different osazones. Principle: Osazone test is a chemical test used to detect reducing sugars. Reagent for this test consists of phenylhydrazine in acetate buffer. Carbohydrates with free or potentially free carbonyl groups react with phenylhydrazine to form osazone. Osazone appears as yellow-colored crystals of characteristic shape, solubility, melting point, and time of formation. 1.4.2.3 Fehling’s Test 1mL of Fehling’s A + 1mL of Fehling’s B. Dilute with 4mL of water. Boil solutions for 1min. Solution should be clear blue. 8 drops of sugar solution -> test tube + 1mL of mixed Fehling’s solution (to each tube) Boil tubes in water bath for 5mins. and note changes. Principle: Fehling’s solution appears deep blue in color and consists of copper sulfate mixed with potassium sodium tartrate and strong alkali. On heating, sample with Fehling’s, bistartarocuprate(II) complex oxidizes the aldoses to corresponding aldonic acids. Copper ions of complex are reduced to insoluble yellow or red-colored precipitate. - If Fehling’s solution is heated in absence of reducing sugars, it forms a black precipitate of cupric oxide. 1.4.2.4 Barfoed’s Test 3mL of Barfoed’s reagent + 10 drops of sugar solution. Place in a boiling water bath for 15mins. Remove and allow to cool spontaneously. After 15mins., observe and record results. Principle: Barfoed’s test is a chemical test used to detect the presence of monosaccharides which detects reducing monosaccharides in the presence of disaccharides. - Barfoed reagent is made up of copper acetate in a dilute solution of acetic acid. - Acidic pH = unfavorable for reduction monosaccharides react in 1-2mins. reducing disaccharides react in 7-8mins. - Once reaction takes place, thin red precipitate forms at the bottom of the sides of the tube. 1.4.2.5 Benedict’s Test  2mL of Benedict’s solution + 5 drops of sugar solution. Place in boiling water bath for 2 mins. Cool and record results. Principle: Enediols reduce cupric ions of Benedict reagent into cuprous ions which are present in the form of insoluble copper oxide or cuprous oxide which is of red color. Red-colored copper oxides get precipitated. - The concentration of reducing sugar in the sample differs from the intensity and shade of the color of the reaction mixture. - greenish -> yellowish -> orange -> brick-red LIPIDS 2.1.1 Saponification of lipids 10g sample lipid -> 250mL erlenmeyer flask. 250mL of 6.0M of Sodium hydroxide + 70mL 95% ethanol Heat steam bath through reflux distillation for 30mins. Shake occasionally. Test for complete saponification - pour 1mL of water to mixture. If cloudiness appears, flask should be heated for another 10-15mins. Principle: It is a chemical reaction where triglycerides react with a strong base, such as NaOH or KOH, to form glycerol and salt of fatty acid(soap) 2.1.2 Separation of Soap by Salting Out 10mL of soap solution + 10-15mL saturated NaCl solution. Filter curdy mass that separates out. Principle: A process that utilizes the principle of decreased solubility of soap in a high ionic strength environment created by adding conc. salt solution. This causes soap to precipitate out of the solution, allowing for its efficient separation and purification from the saponification mixture. 2.1.3 Separation of Fatty Acids and Glycerol 10mL of soap solution + dilute H2SO4 in excess. Test with litmus. Heat at or near boiling point for 2mins.  Note oily layer of fatty acids that form on the surface. Set aside until fatty layer have come to the top. Filter in wet filter paper. Save filtrate for the test for glycerol. Wash fatty acid with hot water 2-3 times until freed from H2SO4 and set. Compare the physical state that of fatty acids prepared from different fats. *Physical state of fatty acids at room temp. in various oils depends on saturation level and specific fatty acid profile of each oil.* 2.1.4 Test for Glycerol (Acrolein Test) Small evaporating dish -> 5 drops of the filtrate (procedure 5 of 2.1.3) + 1g KHSO4 or NaHSO4. Heat over a free flame. Note the odor evolved. 2.1.5 Test for Unsaturation Dissolve 1mL of fat sample in 5mL chloroform. Add iodine drop by drop. Shake sol’n after each drop until color of iodine persists. Note the number of drops of iodine required to give a permanent yellow to orange color. Saponifiable Lipids Triglycerides (olive oil, butter) Phospholipids (lecithin) Waxes Glycolipids (cerebrosides) Non-Saponifiable Lipids Steroids (cholesterol) Terpenes (vit. A, carotenoids) Prostaglandins PLEASE READ BRAIN LIPIDS PART!!!!! 2.2 Brain Lipids Brain tissues - rich in saponifiable and non-saponifiable lipids. These mixture of lipids differ in their chemical composition, and can be isolated from the tissues and from each other through solubility. 2.3 CHARACTERIZATION OF LIPIDS 2.3.1 Salkowski’s Test Small test tube -> 10 drops lipid solution + 20 drops conc. H2SO4 (add down the side of the tube) Observe color change at the interface. - A yellow to brick-red colour is formed indicating the presence of cholesterol.  - There may be a red color in the upper layer(choloroform) and green fluorescence in the lower layer due to heavier sulfuric acid settling. 2.3.2 Liebermann-Burchard Test Small test tube -> 0.5mL lipid solution + 10 drops acetic anhydride. Swirl. Add 4 drops of conc. H2SO4 down the side of the tube. Mix. Note color produced. - Used to detect the presence of steroids. - Deep green color 2.3.3 Molisch Test Small test tube -> 10 drops lipid solution. Evaporate off the solvent from the tube in a boiling water bath. Suspend lipid in 20 drops of distilled water. Add 2 drops of molisch reagent. Mix. Add 20 drops of conc. H2SO4 down the side of the tube. Note the color. 2.3.4 Kraut’s Test Small test tube -> 10 drops of lipid solution. Evaporate off the solvent from the tube in a boiling water bath. Suspend dried lipid in 10 drops of distilled water. Add 15 drops of Kraut’s reagent. Warm tube in water bath for 1-2mins. Note color of sol’n. - Test mainly used for choline due to complexation. - Red-orange solution. 2.3.5 Ninhydrin Test Small test tube -> 10 drops of lipid solution. Add 5 drops of ninhydrin reagent in ethanol. Warm tube in water bath for 1-2mins. Note color of sol’n. Principle: Oxidative deamination followed by decarboxylation and/or condensation. - Test mainly used for cephalins, lecithins, and sphingomyelins. - Uses Nindhydrin reagent (0.1g Nindhydrin in 95% ethanol) - Blue-violet solution indicates a positive result. 2.3.6 Acrolein Test Evaporate off solvent from tube in a boiling water bath. Rub in a mortar about 0.1g of the lipid with equal amount of KHSO4/NaHSO4.  Transfer to a dry test tube cautiously Heat gently at first then strongly. Note the odor. - Is used to detect the presence of glycerol or fat. - Has odor of burnt cooking grease.  PROTEINS Isolation of Gluten: Objective: To isolate gluten from wheat flour. Confirmation of complete removal of starch: Iodine test Amount of insoluble material left compared with the amount of original sample: Gluten constitutes only a portion of the flour ( the rest is composed of: starch, fiber, fat, and other components that are either dissolved or washed away during the isolation process. Composition of Crude Gluten: Gliadin and Glutenin (main types of proteins found in wheat and related grains) Isolation of Bean Protein: Objective: To isolate bean proteins Soaking beans in water: Enhances the extraction process, increases yield, and improves quality of isolated proteins. Used to precipitate mung beans: 1M of Acetic Acid Isoelectric point is around pH 4.5 to 5.5. Predominant proteins present in beans: Storage proteins. Serve as a nutrient source for the seedling during germination. Categorized into two types: Globulins and Albumins. Isolation of Myoglobin from Muscle: Objective: To isolate myoglobin from muscle. Proteins present in Myoglobin from muscle: Oxygen-binding and transport proteins. Serve as a storage site for oxygen and facilitates transport of oxygen within muscle cells, ensuring adequate supply during periods of high metabolic activity. Chemical used in the experiment: 70% buffered-diluted (NH₄)₂SO₄. Isolation of Albumin from Egg White: Objective: Isolate albumin from egg white. Chemicals used: 1N acetic acid and NaCl Hydrolysis of Intact Proteins: Objective: To hydrolyze proteins using acid, alkali, and enzymes Color Clarity Odor Viscosity Acid clear to slightly clearer less intense and less viscous yellowish less unpleasant Alkaline darker, yellow less clear or strong and more viscous to brown more turbid unpleasant, ammonia-like, or putrid Qualitative Test for Proteins Objective: To qualitatively identify the presence of amino acids of the different protein sources in the different test. TEST PRINCIPLE SAMPLE POSITIVE DETECTED/P COLOR OSITIVE SPECIFIC AMINO ACID/S BIURET Detects the presence of peptide bonds in the Peptide Purple sample. The intensity of the color change is bonds directly proportional to the concentration of peptide bonds present in the solution. NINHYDRIN Detects the presence of amino acids. The Amino acids Purple intensity of color change is directly Proline Yellow proportional to the concentration and the type of amino acids present in a solution. XANTHOPROTEIC Detects the presence of aromatic amino Tyrosine Yellow acids. Tryptophan Phenylalanin e MILLON’S Based on the principle of nitrification of the Tyrosine Red phenol group in tyrosine, which then forms complexes with heavy metals. HOPKINS-COLE Layering of concentrated sulfuric acid over a Tryptophan Purple ring mixture of tryptophan-containing proteins with the reagent results in the formation of color at the interface. SAKAGUCHI Reaction between 1-naphthol and the Arginine Red guanidinium groups in arginine in the presence of an oxidizing agent. LEAD ACETATE Detection of sulfur in a solution by the Cysteine Black degradation of the S-H or S-S group in amino acids under strongly alkaline conditions. Denaturation and Precipitation of Proteins: Objectives: To denature proteins using different denaturing reagents; To precipitate proteins using acids, salts of heavy metals, and alcohol. Denaturation - involves the breaking of many of the weak linkages or bonds within a protein molecule that are responsible for the highly ordered structure of the protein in its natural state. Proteins are denatured when its normal shape gets deformed because some of the hydrogen bonds are broken. Could be because of: too much heat is applied when they are exposed to an acid By Acids Relies on changes of a solution pH Addition of acids to solution lowers the pH and leads to positively charging the protein. By Salts of Heavy Metals Addition of salt ions into the solution causes the restriction of the available water molecules for the proteins. By Alcohol Addition of alcohol to the solution reduces the hydration of the protein. ENZYMES Extraction of Amylase and Influence of pH: Materials: Collected washings of Salivary amylase 0.2M NaCl solution 1% starch solution 0.01 iodine solution Buffer solutions Sample: Saliva Enzyme present: Amylase Optimal Temperature: 37°C Optimal pH: 6.8 Substrate: Starch Color reaction of substrate with iodine: Black/Dark Purple/ Dark blue/Blue-black Product: Maltose Color reaction of the product with iodine: Yellow Extraction and Assay of Papain Materials: Papaya fruit 95% ethyl alcohol Ether Assay Materials: Crude papain Cysteine hydrochloride  1% Casein solution 10% cold Trichloroacetic acid (TCA) Function of papain: Break down of proteins Fresh latex is collected from: Young unripe papaya fruit (while it’s still hanging on the tree) How is it collected: By making longitudinal incisions/scratches on the skin about one inch apart using a sharp rod or scalpel. Precipitating agent used: 95% ethyl alcohol Substrate used to measure enzymatic activity: 1% Casein solution pH range optimal for enzymatic activity of papain: pH 5.0-7.0 Activates your papain by reducing disulfide bonds: Cysteine hydrochloride Method used to determine the concentration of papain in the sample: Spectrophotometry Action of Liver Catalase on H₂O₂: Materials: Chicken Liver Hydrogen peroxide Distilled water Sample: Liver/Chicken Liver Enzyme present: Catalase Substrate: Hydrogen peroxide Product/s: Water (H₂O) and Oxygen (O₂) Reaction result: Bubble formation/Effervescence/Bubbling Chemical equation of the reaction: BIO 024: Biochemistry | PTT-based Topic Outline: VITAMINS MEASURING VITAMIN C USING STARCH-IODINE TEST PREPARING THE VITAMIN C EXTRACTS: Procedure: The fruit/vegetable sample is blended and strained in order to extract the vitamin C content. MEASURING VITAMIN C IN THE FOOD SAMPLE: Procedure: The extracted sample is placed in a flask with the starch solution (which serves as the indicator) and acidified with 0.1 M HCl. The sample solution is then titrated with iodine until the endpoint is reached (solution stays blue-black for 15 seconds) The volume of iodine used is equivalent to the vitamin C content of the sample. CHEMICAL REACTION Rationale: Vitamin C is water-soluble and sensitive to heat. Which explains why there is a reduction of vitamin C during cooking. Vitamin C is destroyed with heating and can leach out of vegetables when they're immersed in hot water. APPLICATION: MAGIC WRITING Procedure: 1. Pour 100 ml water into a 500ml-beaker. 2. Add 10 ml of Iodine to the water and stir. 3. Cut (circulate) a section from the notebook paper. 4. The paper must fit inside a 500ml-beaker 5. Squeeze the juice of the lemon/lime into another beaker 6. Dip the art brush into the lemon/lime juice 7. Write a message on the piece of paper. 8. Allow the juice to dry on the paper. 9. Submerse the paper in the iodine solution in the bowl. Rationale Principle is the same as the previous activity. Iodine reacts with the starch present in paper which explains the blue- black/purple color. Vit. C prevents that reaction from occurring hence the absence of blue-black color Samantha Fam Llemit | 1 BIO 024: Biochemistry | PTT-based Alizarin: ○ yellow (pH 6.8). Topic Outline: NUCLEIC ACIDS ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS ISOLATION OF YEAST RNA Materials a.Dried Yeast b.1%NaOH HYDROLYSIS OF YEAST RNA c. Distilled water Rationale: d.Gauze RNA is hydrolyzed or “split” in order to asses its composition. e.Glacial acetic acid f.95% ethanol PROCEDURE: g.concentrated HCl 1. Place a small amount of the yeast RNA in a test tube and h.Ether add 10mL of 10% H2SO4. 2. Place in a boiling water bath for about 30 minutes. Procedure: 3. Perform the following test on the hydrolyzate. RNA is extracted from your yeast by a sequence of steps using different chemicals that enables precipitation and isolation of RNA. TEST FOR PENTOSES The steps involves lysis of yeast, precipitation of unwanted proteins, MOLISCH TEST filtration, precipitation of RNA, and finally washing. PROCEDURE: 1. To 1mLof hydrolyzate add 3 drops of Molisch reagent. If isolation is done well you are left with a clean isolate of RNA, however if 2. Incline the tube. Add on the side of the tube, 2mL of concentrated H2SO4. the isolation step is not well performed interfering substances can cause 3. Observe. Note the color. false positives in your tests. Ex. Unwanted proteins can cause a positive Biuret Test. PHYSICAL AND CHEMICAL PROPERTIES - Test for Carbohydrates MATERIALS a. Cold water Principle: A purple ring forms at the interface when Molisch's b. Hot water reagent and sulfuric acid are added to a carbohydrate solution. c. Ethanol d. 6M HCl e. 6M NaOH f. 10% NaOH solution g. CuSO4 solution BIAL'S ORCINOL TEST OR TEST FOR RIBOSE h. methyl red PROCEDURE: i. congo red 1. To 1mL of Bial’s reagent, add 1mL of hydrolyzate. Mix. j. alizarin 2. Boil gently for 5-10 minutes until bubbles come to the surface SOLUBILITY 3. Note the color obtained. Procedure: Label 5 test tubes and test the solubility of a small pinch of RNA in 1mL of each of the solvents: - Test for Pentoses/ Ribose a. Cold water - Low solubility Principle: When orcinol reacts with pentoses in the b. Hot water - High solubility (depending on the temperature) presence of hydrochloric acid, it produces a c. Ethanol - Low solubility blue-green color. d. HCl - Low solubility e. NaOH. - High solubility The solubility of most solid or liquid solutes increases with increasing temperature. TEST FOR PHOSPHATE BIURET TEST (AMMONIUM MOLYBDATE TEST) Procedure: PROCEDURE: 1. To a small amount of RNA, add 1mL of 10% NaOH and 5 drops of CuSO4 1. To 1mL of hydrolyzate, add 6M NH4OH dropwise until the mixture becomes solution. alkaline to litmus. 2. Mix the solution by slightly inverting the tube. Note the color. 2. Acidify with 6M HNO3. 3. Add 1mL of (NH4)2MoO4 solution. 4. Heat in a water bath. Note the formation of precipitate. - Test to detect peptide bonds. When a mixture containing phosphate is heated with conc. HNO3 and Ammonium Principle: the cupric (Cu+2) ions in the biuret Molybdate solution, a canary yellow precipitate of Ammonium Phosphomolybdate is reagent bind to the nitrogen atoms in the formed. peptide bonds of proteins forming a violet-colored copper complex. PH Procedure: 1. Dissolve a pinch of RNA in a small beaker with 3mL of hot water. 2. Stir with a microstirrer. 3. Place a piece of red litmus paper in a watch glass and touch it with the tip of - Test for Inorganic Phosphate the micro stirrer used in step 2. 4. Divide the RNA solution into 3 different test tubes. Principle: Formation of yellow ppt. via the stated 5. Add 3 drops each of methyl red, congo red and alizarin to test tubes 1 ,2,3 reaction Methyl Red: ○ red (pH 6.2) TEST FOR PURINES PROCEDURE: 1. To 1mL of hydrolyzate, add 6M NH4OH dropwise until the mixture becomes alkaline to litmus. Congo Red: 2. Add a few drops of 1% AgNO3 until a change is noted. ○ blue (pH 5.0). - Test for Purines Principle: Formation of gelatinous ppt. Samantha Fam Llemit | 2 BIO 024: Biochemistry | PTT-based TEST FOR PYRIMIDINE (WHEELER-JOHNSON TEST ) PROCEDURE: 1. To 0.5mL of the nucleic acid solution with the excess of the bromine water until the solution turns yellow. 2. Remove the excess by boiling the solution until it turns light yellow or colorless. 3. Add the excess Ba(OH)2 solution. Test it with litmus paper. 4. Note the solution’s color. - Test for Pyrimidines (Uracil & Cytosine) Principle: This test is based on the reaction of pyrimidines with a specific reagent that leads to a purple color. Samantha Fam Llemit | 3 BIO 024: Biochemistry | PTT-based TEST FOR CREATININE Urine creatinine comes from creatinine of the muscles, a Topic Outline: URINALYSIS waste product formed in the normal breakdown of muscle tissue. URINALYSIS Creatinine reacts with picric acid in the alkaline medium to TEST FOR UREA form a reddish-orange colored complex of creatinine picrate. Urea is the main end product of protein metabolism in man and other animals. The ammonia released from the deamination of Positive: Presence ot reddish-orange color amino acids are never removed through the urea cycle which Negative: Absence of color change occurs in the liver. The urea formed is then carried by the blood to the kidneys where it is excreted. Positive: Brisk effervescence of N2 gas Negative: Absence of effervescence TEST FOR BILE PIGMENTS (SMITH’S TEST) Bile is synthesized in the liver, stored in the TEST FOR URIC ACID gallbladder and is released into the intestinal tract Uric acid is the final product of purine oxidation in the body. It is during digestion. However, in certain pathologic derived from the degradation of nucleoproteins in food and of the conditions the bile is not excreted in the intestine but body cells. accumulates in blood and excreted in the urine. In this test, the uric acid reduces phosphotungstic acid to give a Tests for bile pigments (bilirubin, bilicyanin, biliverdin) blue colored compound. depend upon oxidation of these bile pigments Uric acid is a reducing agent in alkaline medium. It reduced resulting in colored derivatives. phosphotungstic acid to tungsten blue. Positive: Presence of emerald/green layer Positive: Formation of blue color Negative: Absence of green layer Negative: Absence of blue color INDICAN TEST TEST FOR OCCULT BLOOD Indican (Potassium indoxyl sulfate) results from the The presence of red blood cells in the urine may indicate decomposition of the amino acid tryptophan. hemorrhage in the kidney or urinary tract Urine indican when hydrolyzed yields indoxyl which is oxidized The test is based on the peroxidase and the liberated oxygen by ferric ions to indigo blue in the chloroform layer. oxidizes organic substances such as benzidine and guaiac Indican converted to indoxyl with HCl, and the created indoxyl powder forming a green or blue color. converted to indigo blue coloured compound with FeCl3. Positive: Presence of blue ring/layer Positive: Indigo blue in chloroform layer Negative: Absence of blue layer Negative: Absence of color change in chloroform layer TEST FOR KETONE BODIES (GUNNING’S TEST) The ketone bodies are formed in the liver and transported to peripheral tissues. Certain abnormal conditions cause an increase in blood ketone bodies above normal levels and consequently increased in urine. The Gunning's test depends on the reaction of acetone and alcoholic iodine resulting in the formation of iodoform crystals. Positive: Yellowish six-pointed star or six-sided plate TEST FOR GLUCOSE (BENEDICTS TEST) Diabetes mellitus is a condition whereby the body cannot normally utilize sugar and consequently a large amount is excreted in the urine. Benedict's test is based on the fact that in a strong alkaline solution and in the presence of heat, reducing sugars will reduce cupric ions to cuprous oxide. TEST FOR ALBUMIN (EXTON’S TEST) The kidneys, in the filtering process retain large protein molecules in the blood, presence of protein in the urine may indicate a disorder in the filtering mechanism. In this test, the sulfosalicy lic acid precipitates the proteins irreversibly. Negatively charged sulfosalicylic acid neutralizes the positive charge on proteins causing denaturation, and hence precipitation of proteins. Positive: Presence of precipitate Negative: Absence of precipitate Samantha Fam Llemit | 4