Date .............
Name ...................................................................................
Group .................
Instructions and lab report for the practical lesson on biochemistry
Topic:
Examination of urine
Task 1: Qualitative estimation of pathological
components of urine
Perform each of the following reactions with a sample of physiological urine, a sample containing the
relevant pathological component, as well as with the unknown samples of urine.
A. Protein in urine
Principle:
Reagents:
1.
2.
3.
4.
Sulfosalicylic acid dihydrate 200 g/l
Sample of urine with protein
Sample of physiological urine
Unknown samples of urine 1-4
Procedure:
• Test with sulfosalicylic acid: to 1-2 ml of sample add a few drops of aqueous sulfosalicylic acid.
Appearance of turbidity or even precipitate indicates presence of protein. The change is best evaluated
against a black background or a page with printed text.
Semi quantitative evaluation of the sulfosalicylic acid test:
Appearance:
Evaluation
Approximate protein concentration:
Traces
0.05 – 0.1 g/l
Slight turbidity (transparent, a
text behind the tube is legible)
+
0.1 – 0.2 g/l
Opaque turbidity (not
transparent, without flakes)
++
0.5 – 1.0 g/l
Milky turbidity with flakes
+++
2.0 – 5.0 g/l
Cheese-like precipitate
++++
above 5.0 g/l
Opalescence
1
B. Blood and blood pigment in urine
Principle:
Reagents:
1. o-tolidine (3,3’-dimethylbenzidine)
2. Heitz-Boyer’s reagent: colorless reduced phenolphthalein in alkaline medium, stored with
several granules of zinc
3. Acetic acid concentrated
4. Hydrogen peroxide 30 g/l
5.
6.
7.
8.
Ethanol
Sample of urine with blood
Sample of physiological urine
Unknown samples of urine 1-4
Procedure:
• “Benzidine” Test:
Dissolve a few grains of o-tolidine in about 2 ml of ethanol and acidify with concentrated acetic acid
(few drops). Add about 2 ml of hydrogen peroxide (the solution must not turn blue at this stage) and
combine with 1-2 ml of the urine sample. If the solution turns blue or blue-green the test is positive.
• Heitz-Boyer’s Test:
In a test tube combine about 1 ml of urine with equal volume of the Heitz-Boyer reagent. Carefully
overlay with hydrogen peroxide. In the presence of hemoglobin a red-violet ring appears at the
interface.
C. Sugar in urine
Principle:
2
Reagents:
1. Fehling’s solution I:
Copper(II) sulfate cryst. 70 g/l
2. Fehling’s solution II:
Sodium hydroxide 250 g/l
Potassium-sodium tartrate cryst. 350 g/l
3. Sample of urine with glucose
4. Sample of physiological urine
5. Unknown samples of urine 1-4
Procedure:
• Fehling’s test
Prepare a fresh Fehling’s reagent before testing for the presence of glucose. Mix the Fehling’s solution
I (copper sulfate) and II (NaK-tartrate with NaOH) in ratio 1:1. Heat a portion of about 1 ml of the
Fehling’s reagent to boiling – it must not change color. By this way presence of reducing agents is
excluded.
Mix about 1 ml of urine sample with a similar amount of cold (new portion) Fehling’s reagent in a test
tube. Boil in water bath. If the glucose or another reducing compound is present, a green-yellow,
yellow, or even a brick-red precipitate develops. The color depends on the amount of glucose in the
urine sample (turbidity with greenish tint – about 8-15 mmol/l glucose, green ppt. – about 25 mmol/l,
greenish-brown ppt. – about 50 mmol/l, brownish-red ppt – about 100 mmol/l, red ppt – over 150
mmol/l glucose).
D. Ketone bodies:
Principle:
Reagents:
1. Sodium nitroprusside cryst.
2. Sodium hydroxide 100 g/l
3. Glacial (concentrated) acetic acid
4. Lestradet’s reagent:
ammonium sulfate 20 g, sodium carbonate anhydrous 20 g,
sodium nitroprusside 0.2 – 1 g.
5. Sample of urine with ketone bodies
6. Sample of physiological urine
6. Unknown samples of urine 1-4
3
Procedure:
• Legal’s nitroprusside test:
Dissolve few crystals of sodium nitroprusside in a test tube with distilled water.
To about 2 ml of urine add 2 − 3 drops of aqueous solution of sodium nitroprusside and alkalize with 3
drops of NaOH. A red color appears that is caused by creatinine (physiological component of urine).
Divide the colored solution into two parts. Add a few drops of glacial acetic acid into one part of
solution: if the color changes to yellow it was caused by creatinine. In contrast, in the presence of
ketone bodies the red color turns to red-violet upon addition of the acetic acid.
• Lestradet’s test:
Place a circle of filter paper onto a watch glass and wet it with distilled water. Put a small amount of
Lestradet’s reagent (powder) on the filter paper and add 1 − 2 drops of urine. A purple color
developing within 1 minute indicates presence of ketone bodies.
E. Examination of urine with polyfunctional diagnostic strips
Perform the examination with diagnostic strips only with the unknown samples of urine.
Principles of diagnostic strip tests for leukocytes and nitrite:
Reagents:
1. Polyfunctional strip PHAN (Erba-Lachema Diagnostica s.r.o.)
2. Unknown samples of urine 1-4
Procedure:
Immerse the strip into urine for 2-3 seconds. Wipe edge of the strip against rim of the test tube to
remove excess urine. Wait 60 seconds (120 sec. for leukocytes) and compare the test zones on the strip
with the color scale on the tube label.
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Results:
1) Test tube reactions with the samples of physiological urine and the samples of urine containing the
known pathological components:
Sample
Observed result
Test with sulfosalicylic acid
Urine with protein
Physiological urine
“Benzidine” test
Urine with blood
Physiological urine
Heitz-Boyer’s test
Urine with blood
Physiological urine
Fehling’s test
Urine with glucose
Physiological urine
Legal’s test
Urine with ketone bodies
Physiological urine
Lestradet’s test
Urine with ketone bodies
Physiological urine
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2) Examination of the unknown samples of urine with test tube reactions and the polyfunctional
diagnostic strips:
Sample 1
Test tube
reactions
pH
Dg. strip
Sample 2
Test tube
reactions
Dg. strip
Sample 3
Test tube
reactions
Dg. strip
Sample 4
Test tube
reactions
×
×
×
×
Leukocytes
×
×
×
×
Nitrite
×
×
×
×
Bilirubin
×
×
×
×
Urobilinogen
×
×
×
×
Dg.
strip
Protein
Hemoglobin
Glucose
Ketone bodies
Evaluation and conclusion:
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Task 2: Simple chemical reactions in urine used in
screening of inborn errors of metabolism
A. Fölling’s test with ferric chloride
Principle:
Reagents:
1. Solution of FeCl3 . 6 H2O (store in a dark and tightly closed bottle) 100 g/l
2. Sample of urine with phenylpyruvate
3. Sample of physiological urine
Procedure:
Take about 1 ml of urine with phenylpyruvic acid; add 2-3 drops of FeCl3 solution and mix. A positive
result manifests as a blue-green color. Perform the test with physiological urine as well.
B. Brand’s test with sodium nitroprusside
Principle:
Reagents:
1. Sodium hydroxide 80 g/l
2. Sodium nitroprusside 200 g/l
3. Sample of urine with cysteine
4. Sample of physiological urine
Procedure:
Take about 1 ml of urine with cysteine and alkalize with 3-4 drops of NaOH, add 1-2 drops of sodium
nitroprusside and mix. In case of positive test a cherry red color develops that later disappears.
Perform the test with physiological urine as well.
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C. Test with 2,4-dinitrophenylhydrazine
Principle:
Reagents:
1. 2,4-dinitrophenylhydrazine 1g/l in HCl 2 mol/l
2. Sodium hydroxide 80 g/l
3. Sample of urine with keto acids
4. Sample of physiological urine
Procedure:
Measure 2 drops of urine with keto acids, add the same volume of 2,4-dinitrophenylhydrazine and
leave standing for 5 minutes. Then add 3 drops of NaOH and mix. A positive result is seen as a
brownish-wine color that does not disappear after mixing. Perform the reaction simultaneously with
sample of urine with keto acids and physiological urine, and compare the results.
Results:
Test:
Observation:
Physiological urine
Urine containing the tested
substance
Fölling’s test
(phenylpyruvate)
Brand’s test
(cysteine)
2,4-dinitrophenylhydrazine
(keto acids)
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Task 3: Demonstration of semiautomatic reflectance
photometer for objective semi-quantitative analysis of
urine with diagnostic strips
Principle:
Reagents and equipment:
1. LAURA Smart reader
2. Diagnostic strips for LAURA Smart
3. Urine samples
Result:
Task 4: Measurement of the relative specific gravity of
urine with urinometer
Procedure:
A suitable cylinder is filled with urine sample. Urinometer is dipped to the urine and the relative
specific gravity of the sample is read from its scale.
This measurement will be demonstrated by your instructor.
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Task 5: Examination of urinary sediment in phase
contrast
Reagents:
Authentic sample of fresh urine – infectious material
Procedure:
A fresh sample of urine is centrifuged at 400 – 600 g for 10 minutes at 10 °C and kept on ice. Majority
of supernatant is removed and the urinary sediment is resuspended in the remaining small volume of
supernatant. A microscopic preparation made from the concentrated urinary sediment is examined
under microscope with phase contrast.
Results and conclusion:
Describe the findings in the preparation of urinary sediment
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