LAB TESTS SPOTS-JL biochemistry ka bhaang bhosda.pptx

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1. LAB TESTS SPOTS - JL 6/8/22 1

2. 1 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 2

3. 1 - KEY A) Molisch’s test - Molisch’s test is a sensitive but non-specific test and is given positive by all types of carbohydrates. If the oligosaccharides or polysaccharides are present they are first hydrolyzed to mono saccharides which are then dehydrated to give the test positive. B) Lead acetate test - Lead acetate test is is given positive by sulphur containing amino acids (cysteine or cystine) or proteins containing these amino acids. Methionine does not give this test positive, since the sulphur in methionine is not split by alkali. The presence of cystine in urine is often indicative of amino acid reabsorption defects. 6/8/22 3

4. 2 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 4

5. 2 - KEY A) Barfoed’s test - Barfoed’s test is given positive by reducing monosaccharides. Barfoeds test helps to distinguish monosaccharides from disaccharides. On prolonged heating disaccharides can also give this test positive, h ence , the solution should be boiled for 3 minutes only. B) Biuret test - Biuret test is given positive by all proteins, polypeptides and oligopeptides. Dipeptides and amino acids do not give this test positive. The minimum requirement for this test to be positive is 2 peptide bonds. 6/8/22 5

6. 3 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 6

7. 3 - KEY A) Benedict’s test - Benedict’s test is a semi quantitative test for reducing sugars. The color of the precipitate gives a rough estimate of the reducing sugar present in the sample . Green color - Upto 0.5 gm%(+), green precipitate - 0.5 to 1.0 gm%(++) and yellow precipitate - 1.0 to 1.5 gm%(+++). B) Ninhydrin test - Ninhydrin test is done to detect the presence of alpha amino group of proteins and alpha amino acids. It is one of the most sensitive tests and is given positive by proteins, peptones, peptides, amino acids, ammonia and other primary amines. It can be used qualitatively (e.g. for chromatographic visualization) or quantitatively (e.g. for peptide sequencing). 6/8/22 7

8. 4 Identify and state the significance of tests (A) and (B) ( A) ( B)

9. 4 - KEY A) Seliwanoff’s test - Seliwanoff’s test is given positive by ketohexoses. I t is answered by fructose, sucrose and other fructose containing carbohydrates. This test distinguishes between glucose and fructose . Over heating of the solution should be avoided. Upon continuous boiling, aldoses get converted to ketoses and give a positive reaction. B) Heat coagulation test - Heat coagulation test is employed to detect the presence of albumin in urine. When heated at iso electric point, some proteins undergo irreversible denaturation to produce thick coagulum. Albuminuria is seen in severe dehydration, ascites, heart disease, fever, nephrotic syndrome, pyelonephritis, glomerulonephritis, renal tumours and urinary tract infections. 6/8/22 9

10. 5 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 10

11. 5 - KEY A) Millon’s test - Millon’s test is a confirmatory test for tyrosine. Phenolic amino acids like tyrosine and its derivatives give this test positive. Proteins containing tyrosine give this test positive. Tyrosine is excreted in the urine in tyrosinemia. B) Xanthoproteic test - Xanthoproteic test is a confirmatory test for aromatic amino acids. Nitration of phenyl alanine under these conditions normally does not take place, hence this test is not answered by phenylalanine . Thus the amino acids with phenol (tyrosine ) and indole (tryptophan ) ring give this test positive, while the amino acids with phenyl (phenylalanine ) ring does not give this test positive. 6/8/22 11

12. 6 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 12

13. 6 - KEY A) Hopkin Cole test - Hopkin Cole test is a confirmatory test for tryptophan. All proteins containing tryptophan give a positive reaction to this test. The reaction is due to the presence of indole ring of the amino acid tryptophan. Tryptophan is found in the urine in Hartnup disease (a disorder of amino acid transport). B) Picric acid test - Picric acid test is a test for precipitation of proteins by acidic reagent. Appearance of yellow precipitate indicates presence of proteins. Proteinuria is seen in severe dehydration, ascites, heart disease, fever, nephrotic syndrome, pyelonephritis, glomerulonephritis, renal tumours and urinary tract infections. 6/8/22 13

14. 7 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 14

15. 7 - KEY A) Phosphotungstic acid reduction test - Phosphotungstic acid reduction test is done for the detection of uric acid in the urine. Uric acid is a reducing agent in alkaline medium. It reduces phosphotungstic acid to tungsten blue. Increased urinary uric acid is seen in gout, acute renal failure, Fanconi’s syndrome, cancers , leukemia, administration of ACTH and Wilson's disease. B) Jaffe’s test - Jaffe’s test is done for the detection of creatinine in the urine. Creatinine reacts with picric acid in the alkaline medium to form a reddish colored complex of creatinine picrate. Increased urinary creatinine is seen in renal failure and muscular disorders. 6/8/22 15

16. 8 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 16

17. 8 - KEY A) Isoelectric pH precipitation test - Isoelectric pH precipitation test is performed with casein. Proteins have minimum solubility at their isoelectric pH. Proteins are easily precipitated from their solution by adjusting the pH close to their isoelectric point by addition of acid or alkali. Appearance of green color indicates that the pH is close to 4.6 which is the isoelectric pH of casein. B) Rothera’s test - Rothera’s test is done for the detection of ketone bodies in urine. Appearance of purple ring indicates the presence of ketone bodies in urine . Rothera’s test is given positive by acetone and acetoacetate. Beta hydroxy butyrate is not detected by Rothera's test. Causes of ketonuria are diabetes mellitus and starvation. 6/8/22 17

18. 9 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 18

19. 9 - KEY A) Sodium hypobromite test - Sodium hypobromite test is done for the detection of urinary urea. When urea is treated with sodium hypobromite, it decomposes to give nitrogen gas (effervescence). Increased urinary urea is seen in fever , renal diseases, diabetes mellitus and excess of adrenocortical activity. B) Sakaguchi test - Sakaguchi test is used for the detection of free arginine or arginyl residues in proteins. Free a rginine or arginyl residues in proteins react with alpha naphthol and alkali hypobromite to give carmine red colored complex. Arginine is present in the urine in argininemia (a disorder of urea cycle caused due to deficiency of the enzyme arginase). 6/8/22 19

20. 10 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 20

21. 10 - KEY A) Sulfosalicylic acid test - Sulfosalicylic acid test is done for the detection of proteins in urine . Negatively charged sulphosalicylic acid neutralizes the positive charge on proteins causing denaturation, and hence precipitation of proteins. Proteinuria is seen in severe dehydration, ascites, heart disease, fever, nephrotic syndrome, pyelonephritis, glomerulonephritis, renal tumours and urinary tract infections. B) Fouchet’s test – Fouchet’s test is done for detection of bilirubin in urine. Appearance of green precipitate on the filter paper on addition of Fouchet’s reagent indicates presence of bilirubin in the urine. Bilirubin is present in the urine in obstructive jaundice. 6/8/22 21

22. 11 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 22

23. 11 - KEY A) Inversion test - Inversion test is a confirmatory test for sucrose. Sucrose is a non-reducing sugar, since it does not have free aldehyde or ketone group to cause reduction, hence it gives a negative reaction with Benedict’s reagent. But upon boiling with HCl, sucrose is hydrolyzed to yield glucose and fructose, which give positive Benedict’s test. B) Glucosazone - Glucose forms characteristic needle shaped osazone crystals . Glucose, fructose and mannose produce the same osazone because of the similarities in their molecular structure. A solution of reducing sugar when heated with phenyl hydrazine forms characteristic yellow crystalline compounds called osazones. Osazones help in identification of the sugars. 6/8/22 23

24. 12 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 24

25. 12 - KEY A) Maltosazone - Maltose forms characteristic star/sunflower shaped osazone crystals . A solution of reducing sugar when heated with phenyl hydrazine forms characteristic yellow crystalline compounds called osazones. Osazones help in identification of the sugars. B) Lactosazone - Lactose forms characteristic powderpuff shaped osazone crystals . A solution of reducing sugar when heated with phenyl hydrazine forms characteristic yellow crystalline compounds called osazones. Osazones help in identification of the sugars. 6/8/22 25

26. 13 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22

27. 13 - KEY A) Half saturation test - Albumin gives positive biuret test after half saturation. Albumin solution is mixed with ammonium sulphate solution. This is filtered and biuret test is performed on filtrate. Positive biuret test indicates that albumin is not precipitated by half saturation with ammonium sulphate. Albumin, since it tenaciously holds on to water, needs a much higher concentration of salt than globulins to be precipitated. B) Full saturation test - Albumin gives negative biuret test after full saturation. Albumin solution is mixed with solid ammonium sulphate powder until it is saturated. This is filtered and biuret test is performed on the filtrate. Negative biuret test indicates that albumin is precipitated by full saturation with ammonium sulphate . 6/8/22 27

28. 14 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 28

29. 14 - KEY A) Trichloro acetic acid test - TCA precipitates proteins . Negatively charged trichloroacetic acid is a protein precipitant. TCA lower the pH of the medium, when proteins carry net positive charges. These protein cations are electrostatically complexed with negatively charged ions and thick flocculent precipitates are formed. In the clinical laboratory TCA is used for precipitating proteins. B) Mercuric nitrate test - Mercuric nitrate precipitates proteins . When solution of mercuric nitrate (heavy metal salt) is added to protein in alkaline solution, the cations interact with the negatively charged groups on the proteins causing precipitation as metal proteinate complexe . Based on the same principle, raw egg white is used as an antidote in mercury poisoning. 6/8/22 29

30. 15 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 30

31. 15 - KEY A) Silver nitrate test - Silver nitrate test is done for the detection of urinary chlorides. Silver chloride is precipitated in the presence of nitric acid and silver nitrate. Chlorides are present in normal urine. Urinary chlorides are increased in polydipsia and Addison's disease. B) Barium chloride test - Barium chloride test is done for the detection of urinary sulphates . Sulphate is precipitated as barium sulphate with barium chloride. Sulphates are present in normal urine. Urinary sulphates are increased in cystinuria , homocystinuria, obstructive jaundice, hepatocellular jaundice and cyanide poisoning. 6/8/22 31

32. 16 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 32

33. 16 - KEY A) Potassium oxalate test - Potassium oxalate test is done for detection of urinary calcium. With potassium oxalate in acidic medium, calcium is precipitated as calcium oxalate. Calcium is present in normal urine. Increase in urinary calcium is seen in hyperparathyroidism , hyperthyroidism, hypervitaminosis D, multiple myeloma, renal stones and renal tubular acidosis. B) Ammonium molybdate test - Ammonium molybdate test is done for detection of urinary inorganic phosphate . Upon warming with ammonium molybdate in the presence of HNO 3 , inorganic phosphate is precipitated as canary yellow ammonium phosphomolybdate. Phosphate is present in normal urine. Increase in urinary phosphate is seen in rickets , osteomalacia, hyperparathyroidism and acidosis . 6/8/22

34. 17 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 34

35. 17 - KEY A) Ammonia test - Ammonia acid test is done for the detection of ammonia in urine . Ammonia is evaporated when urine is made alkaline and turns the litmus paper blue. Urinary ammonia is derived from glutamine and other amino acids. Ammonia is excreted in normal urine. Increased urinary ammonia is seen in diabetic keto acidosis, ingestion of acid forming foods, excessive water ingestion and urinary tract infections. B) Heller’s test – Heller’s test is done for detection of proteins in urine. Concentrated HNO 3 causes denaturation and hence precipitation of proteins. Proteinuria is seen in severe dehydration, ascites, heart disease, fever, nephrotic syndrome, pyelonephritis, glomerulonephritis, renal tumours and urinary tract infections. 6/8/22 35

36. 18 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 36

37. 18 - KEY A) Gerhardt’s test - Gerhardt’s test is done for the detection of acetoacetate in urine . This test is not given by acetone and beta hydroxy butyrate . Causes of ketonuria are diabetes mellitus and starvation. B) Ehlrich’s test - Ehlrich’s test is done for detection of urobilinogen in urine. Urobilinogen is found in urine in hepatic and prehepatic jaundice. It is present in excessive amount in prehepatic jaundice and is completely absent in post hepatic jaundice. An increased urobilinogen concentration in urine is a sensitive index of liver dysfunction or hemolytic disorders. 6/8/22 37

38. 19 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 38

39. 19 - KEY A) Hays sulfur test - Hays sulfur test is done for the detection of bile salts in the urine . Bile salts lower the surface tension allowing the sulphur powder to sink. Bile salts are present in the urine in obstructive jaundice. B) Tolidine test - Tolidine test is done for detection of blood in urine. Causes of g ross hematuria (urine appears reddish) are renal stones, malignancies, trauma, tuberculosis and acute glomerulonephritis . Causes of microscopic hematuria (blood is not visible to naked eyes) are malignant hypertension, sickle cell anemia, coagulation disorders, polycystic kidney disease, incompatible blood transfusion & auto immune hemolytic anemia. 6/8/22 39

40. 2 Identify and state the significance of tests (A) and (B) ( A) ( B) 6/8/22 40

41. 2 0 - KEY A) Gmelin’s test - Gmelin’s acid test is done for the detection of bile pigments in urine . Play of colors (yellow, red, violet, blue and green) indicates positive test. Bile pigments are oxidised to various chemical products. Nitric acid is used as the oxidizing agent. Bilirubin is present in the urine in obstructive jaundice. B) Benzidine test - Benzidine test is done for detection of blood in urine. Causes of gross hematuria (urine appears red) are renal stones, malignancies, trauma, tuberculosis & acute glomerulonephritis. Causes of microscopic hematuria (blood is not visible to naked eyes) are malignant hypertension, sickle cell anemia, coagulation disorders, polycystic kidney disease, incompatible blood transfusion & auto immune hemolytic anemia. 6/8/22 41