Experiment #13: Urine

I. Objectives

At the end of the experiment, the students should be able to:

a. explain the pathological constituents of urine; and

b. define the test that will detect the presence of different substances present in urine.

II. Materials

Beaker, Urinometer, Dropper, Test Tubes, Graduated Cylinder, Bunsen Burner, Water Bath

III. Reagents

Benzidine Reagent (Benzidine in Glacial Acetic Acid), 3% Hydrogen Peroxide, Acetic Acid, conc. Nitric
Acid, Sodium Nitroprusside, Fehling’s A and B, conc. Ammonium Hydroxide

IV. Procedures

I. Qualitative Tests for Constituents of Urine

A. Physical Properties

1. Collect and measure total volume of urine sample. Note the color.
2. Determine the transparency or cloudiness of the sample.
3. Determine the specific gravity by using urinometer.
4. pH – determine the approximate pH by dipping a strip of litmus paper into the urine.

B. Chemical test for normal constituents of urine shows:

a. Anions like chlorides, sulfates, and phosphates

b. Cations like sodium, potassium, ammonium, calcium, and magnesium

C. Chemical test for pathologic or abnormal constituent of urine

The following constituents, protein, sugar, ketone bodies, and bile pigments, are found in normal urine
in small quantities, too small to be detected by ordinary laboratory procedures. They become pathologic when
excreted in abnormal amounts. Blood, however, when present in urine (except menstrual blood) is always
pathologically significant.
II. Pathological Urine

A. Sugar

1. Place 10 drops each of Fehling’s A and B in a test tube then add 10 drops of urine sample.
2. Heat in boiling water for two minutes.
3. Observe a color change.

B. Blood

1. To 1 mL of freshly prepared benzidine solution, add 10 drops of urine sample.

2. Add 6 drops of 3% hydrogen peroxide. Take note of the result.

C. Protein

1. Heat and Acetic Acid Test

a. Fill a test tube about ¾ full of urine.
b. Heat the upper portion gently to boiling for 1-2 minutes being careful not to shake the tube.
c. Rotate the tube to prevent cracking. Turbidity may be due to albumin, phosphates or carbonates.
d. Add 3 drops of acetic acid drop by drop, boiling between each drop. If turbidity disappears, it is due to
carbonates and phosphates. A faint trace of albumin may appear upon boiling and increase upon
addition of acetic acid.
2. Heller’s Nitric Acid Test
a. Place 1 mL urine sample in a test tube.
b. Hold the test tube in an inclined position and carefully pour 10 drops of conc. Nitric Acid down the side
of the test tube.
c. Note the reaction at the zone of contact between the solutions.

D. Ketone Bodies

1. To 1 mL urine sample, add a few drops of freshly prepared sodium nitroprusside. Shake.
2. Add 1 mL of conc. ammonium hydroxide carefully down the side of the tube to form a layer over urine.
3. Observe and note the color reaction.
 Experiment 13

Urine

Name: Date:

Section/Group: Instructor:

I. Qualitative Tests
A. Physical Properties
1. Color

2. Specific Gravity

3. pH

4. Transparency

B. Chemical Tests

Give the confirmatory test for:

1. Anions
a. Chloride
b. Sulfate
c. Phosphate
2. Cations
a. Sodium
b. Potassium
c. Calcium
d. Magnesium

C. What test will detect the presence of the following substance in urine and what clinical conditions result
from them?

a. Protein

b. Sugar

c. Ketone Bodies
 d. Blood

e. Bile Pigments

II. Observations

A. SUGAR

B. BLOOD

C. PROTEIN
I. HEAT AND ACETIC ACID TEST

II. HELLER’S NITRIC ACID TEST

D. KETONE BODIES
III. Questions/Exercises

1) Distinguish between hematuria and hemoglobinuria.

2) What is the clinical importance of bilirubinuria? Or urobilinogenuria?

3) How will you differentiate glucosuria from lactosuria? What is the clinical importance?