Activity #2

PHYSICAL EXAMINATION OF THE URINE

Objectives:
At the end of the activity the students shall be able to:
1. Perform physical and macroscopic determine the urine in terms of color, transparency, volume and specific gravity
2. Demonstrate the proper use of urinometer and refractomer
3. Differentiate between refractometer and urinometer in terms of their advantages and disadvantages
4. Mention the effect of changes in temperature while performing urinometer and calculate techniques for correcting
values
5. Distinguish normal test results in the urine physical examination
6. Compare abnormal macroscopic test results with pathologic conditions
7. Relate quality assurance in the physical examination of urine
8. Describe correct test results using standard format of reporting

Materials:
- Test tube
- Urinometer
- Refractometer
- Dropper
- Urine

A. Color and Transparency/ clarity

Procedure:
1. Mix the specimen and place 12 ml urine in a test tube
2. Examine the specimen under a good light source, looking through the urine against white background
3. Determine color and clarity

Color: Normal= straw to amber

Other description: light yellow, yellow, dark yellow, amber

Transparency/ appearance/ clarity

Normal= clear when voided but may become turbid upon standing
Other description:
Clear: transparent; no particle seen
Hazy: print easily seen through urine; few particles seen
Cloudy: print blurred urine; many particles seen
Turbid: print cannot be seen through urine
Milky: May precipitate or be clotted
Note: Degree of turbidity should correspond with the amount of materials observed under the microscope

B. Specific Gravity
- The density of a substance compared with the density of water at specified temperature
Methods:
1. Urinometry- uses urinometer or hydrometer adapted to measure the specific gravity at room temperature
 PI: water displacement or buoyancy
Disadvantage: requires 10-15 ml

Accuracy may be checked by measuring the specific gravity of

a. Distilled water- specific gravity should be 1.000
b. Potassium sulfate- specific gravity should be 1.015

Procedure:
a. Pour the specimen into the urinometer tube or cylinder until it is about ¾ full
b. Insert the urinometer with a spinning motion to make sure that it is floating freely (be sure that it does not touch the
sides or bottom of the cylinder)
c. Read the bottom of the meniscus
Note: Insufficient amount of sample or sp gravity greater than the scale can be diluted and retested. Multiply the decimal
factor by the dilution factor to get the actual specific gravity reading

Reading is affected by;

A. Temperature
- Subtract 0.001 from the reading for every 3 degrees centigrade that the specimen temperature is below the urinometer
temperature
- Add 0.001 to the reading for every 3 degrees centigrade that the specimen temperature is above the urinometer
temperature
B. Glucose
- Subtract 0.004 for every gram of glucose/dl of urine
C. Protein
- Subtract 0.003 for every gram of protein/dl of urine
2. Refractometry
- Measurement of light- bending capability of solutions
- Refractometer determines the concentration of dissolved particles in a specimen by measuring refractive index.
Refractive index is the comparison of the velocity of light in air with the velocity of light in a solution.
- Also known as Total Solid Meter

Procedure:
1. Clean the surfaces of the cover and prism with a drop of distilled water and a damp cloth and allow it to dry
2. Apply a drop of urine at the notched bottom of the cover so that it flows over the prism surface by capillary action
3. Read directly on the specific gravity scale (the sharp dividing line between light and dark contrast)

Refractometer may be calibrated using the following:

Sp gravity reading
a. Distilled water -1.000
b. 5% Na Cl -1.022
c. 9% sucrose -1.034
Note: The specific gravity reading on the refractometer is generally slightly lower than a urinometer reading by about 0.002.
Advantages:
 1. Uses small amount of urine (1-2 drops)
2. No temperature correction are required
3. Simple to operate
4. Gives reliable results
3. Reagent strip (Dipstick)
Principle: based on pK change of polyelectrolytes in relation to ionic concentration of urine
Color changes from blue (1.000) through shades of green, to yellow (1.030). Color chart provided indicates values of 1.000
to 1.030 in increments of 0.005

3 main ingredients in the reagent are:

1. Polyelectrolytes
2. Indicator ( bromthymol blue)
3. Buffer
NAME: ______________________________________________SECTION: ______________ DATE: _________

PHYSICAL/ MACROSCOPIC EXAMINATION OF URINE

GROUP RESULT NORMAL RESULT
COLOR
TRANSPARENCY
SPECIFIC URINOMETER
GRAVITY REFRACTOMETER
REAGENT STRIP

Steps in reagent strip reading

1. Draw the results of macroscopic examination of urine

Color Transparency
 2. Draw a urinometer and label its parts

3. Draw a refractometer and label the parts

4. Give the advantages and disadvantages of the following in measuring the specific gravity of urine

METHOD ADVANTAGES DISADVANTAGES

URINOMETER
REFRACTOMETER
REAGENT STRIP

5. Discuss the proper procedure of collecting a 24 hr. urine specimen for quantitative analysis
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6. Give the pathologic and non-pathologic causes of a turbid urine
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7. What is the importance of indicating of the color and transparency of the urine?
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8. How important macroscopic examination of urine in diagnosing of disease

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 Activity #3
CHEMICAL EXAMINATION OF THE URINE

OBJECTIVES: At the end of the activity the students can able to:

1. Describe the principle and sources of errors involved in reagent strip testing
2. Determine the proper technique for reagent strip testing
3. Accomplish chemical analysis using the following manual methods
a. Benedicts test for sugar
b. Heat and acetic test for protein
c. Picric acid test for protein
d. Sulfosalicylic acid for protein
4. Understand and report correct test results for the reagent strip and manual methods
5. Differentiate between normal and abnormal chemical results

MATERIALS:

Per group: Per class

Reagent strip Acetic Acid
Reagent strip Benedict’s reagent
Pipette Picric acid
Aspirator
Litmus Paper

Reagent strip test:

-give a simple, rapid way for performing chemical analysis of urine including ph, protein,
glucose, bilirubin, ketones, urobilinogen, nitrite, leukocytes and specific gravity.

Reagent strips- plastic strips that contain one or more chemically impregnated test sites on
an absorbent pad.

General Procedure for urine reagent strip testing

Procedure:
1. Dip the reagent strip briefly into a well-mixed urine
2. Remove excess urine from the reagent strip by touching the edge of the strip of the
strip to the tube as the strip is withdrawn
3. Hold the strip horizontally, wait for the reading time specified by the manufacturer
4. Compare the color of dipped reagent strip with the color chart provided.

CHEMICAL TESTS PERFORMED IN ROUTINE URINALYSIS

A. Reaction/Ph= reflects the ability of the kidney to maintain normal hydrogen ion
concentration in plasma and extracellular fluid
 Normal value: 1st morning= 5.0 -6.0
Random = 4.5 – 8.0

Methods:
1. Reagent strip (pHrange )
Principle is based on double indicator system of methyl red and bromthymol blue. Color
change from orange at pH5 through yellow and green to deep blue at pH 9.

B. PROTEIN
NV: less than 10mg/dl or 100 mg/24 hrs.

METHODS:
1. Acid precipitation methods- detects all protein (albumin and globulin)
a. Heat and acetic acid
PI: based on the precipitation of protein by heat and coagulation by chemical
agents

Procedure:
a. Place about 5 ml filtered urine in a test tube
b. Heat upper (2 cm) portion of urine and observe for cloudiness (maybe due
to phosphates & albumin)
c. Add 2 or 3 drops of 30% HAc (cloudiness due to phosphates will disappear

d. Repeat the heating and adding of Acetic acid twice in order to make the
specimen sufficiently acid.

Note: A persistent cloudiness indicates albumin

Result:

(-) no cloudiness or haziness

(+) diffuse cloud
(++) granular cloud
(+++) distinct flocculation
(++++) large flocculation dense, sometimes solid

2. Sulfosalicylic Acid test (Exton’s)

- There’s no need to heat

Reagents:
Sulfosalicylic acid 50 gm
Sodium Sulfate anhydrous 88 gms
If hydrated 200 gms
Dilute with distilled H2O up to 1 liter

Procedure:
a. Place 3 ml of centrifuged urine in a test tube
b. Add equal amount of SSA
c. Invert to mix. Let stand for exactly 10 min
 d. Invert again twice
e. Observe the degree of precipitation and grade the result

Positive Result:
Negative no increase in turbidity
Trace noticeable turbidity
1+ distinct turbidity with no granulation
2+ turbidity and granulation with no flocculation
3+ turbidity with granulation & flocculation
4+ clumps of precipitated protein or solid ppt

3. Heller’s Ring or Heller’s Heat or nitric acid test

Procedure:
a. Place 2 ml of urine in a test tube
b. Overlay it with 2 ml of conc. Nitric acid
Positive Result:
White opaque ring at the zone of contact

4. Picric Acid test

Procedure:
a. Filter urine
b. Place 2.5 ml urine in a test tube
c. Add equal amount of saturated solution of picric acid
Positive result:
Light cloudiness to a heavy flocculation depending on amount of albumin present
5. Reagent strip technique (dipstick)
Test is based on the principle of protein error of indicators

Interpretation of results:

Negative = no change in color (yellow)

Positive = yellow color changes to a yellow green, to green, to blue
green depending on the concentration of protein

Manner of reporting

Trace, 1+, 2+, 3+, 4+

 Name: _____________________________________ Date performed: _________
Section:____________________________________

Routine chemical examination of Urine (Protein)

Methods:
1. Heat and acetic acid _______________
2. Hellers’ ring test _______________
3. Picric acid _______________
4. Reagent strip _______________

1. What are the causes of the early deterioration of reagent strips? Enumerate ways how
to avoid them.

2. List sources of error/interference that may occur with the following test of urine
protein: false positive and false negative.
 Activity #3-B
CHEMICAL EXAMINATION OF THE URINE
(GLUCOSE)

METHODS:
1. Copper Reduction Tests
A. Benedict’s Test
Principle: Glucose in urine reduces the blue alkaline copper sulfate precipitate. In this method, the Benedict’s
reagent must first be tested before use by heating the reagent. A change in color from the original blue means
the solution has already undergone reduction and cannot be used anymore.

Benedict’s reagent
Copper sulfate 17.3 gms
Na citrate 173.0 gms
Na carbonate (anhydrous) 100 gms
Dilute with water up to 1 liter
Procedure:
a. Place 5 cc Benedict’s reagent in a test tube
b. Add 8-10 drops of urine, mix and boil for 2 minutes over a flame or boil for 5 min in a water bath
c. Read the result at once

Interpretation:
Negative clear blue
Trace green w/ no precipitation
1+ green w/ yellow precipitate
2+ yellow or yellow green with yellow precipitate
3+ muddy orange with yellow precipitate
4+ orange to red precipitate

B. Clinitest
-copper Reduction Tablet Test
Each tablet contains copper sulfate, sodium hydroxide, sodium carbonate and citric acid. Copper sulfate reacts
with reducing substances in the urine converting cupric sulfate to cuprous oxide.
Involves pass through phenomenon if sugar in the urine is more than 2 gm/dl. Here, the solution goes
through the entire range of colors and back to a dark greenish brown color.

Procedure:

Five Drop Method

a. Place 5 drops of urine in a test tube
b. Add 10 drops of water
c. Add one clinitest tablet (do not shake or touch the bottom of the tube, it is hot. (heat is caused by reaction of
NaOH with water and citric acid)
d. Wait for 15 seconds after boiling stops, then shake the tube gently and compare the result with the color scale
immediately.
 2. Reagent strip- specific test for glucose
a. Employs the glucose oxidase method by impregnating the testing area with glucose oxidase, peroxidase,
chromogen and buffer to produce a double sequential enzyme reaction.
Reagent strips differ in chromogen used
Multistix - potassium iodide chromogen, color changes from blue to brown in 30 seconds
Chemstrip - Aminopropyl-carbazol chromogen
Color changes from yellow to orange brown in 60 seconds
Clinistix - O toluidine chromogen
Color changes from pink to purple

Glucose+ O2 –Glucose oxidase-----→ gluconic acid + H2O2

H2O2 + chromogen ---Peroxidase-------→ oxidized chromogen +H2O

 Activity #4
MICROSCOPIC EXAMINATION OF THE URINE

OBJECTIVES: At the end of this activity, the students shall:

1. Apply the correct techniques in focusing specimen under the microscope
2. Demonstrate the proper skills in the microscopic urine examination including specimen preparation, centrifugation,
sediment preparation and reporting of results
3. Identify the microscopic elements seen in normal and abnormal urine samples
4. Correlate microscopic findings in its appropriate format

Care of the microscope:

1. Carry microscope with two hands. Take hold of the handle with one hand, and the other hand supporting the base
2. Always hold the microscope in vertical position
3. Clean the lenses with lens paper. Clean the oil immersion lens after each use
4. Never leave the slide on the stage of the microscope nor on the table after examination
5. Store the microscope with low power objective in position and the stage centered.

MATERIALS:
Slides & coverslips

BRIGHTFIELD MICROSCOPY OF UNSTAINED URINE

Subdued light is needed to delineate the more translucent formed elements of the urine such as hyaline casts, crystals
and mucus threads.
Procedure:
1. Mix the urine thoroughly and place about 10-15 ml in a centrifuge tube.
2. Centrifuge for 5 minutes at 1500 to 2500 rpm or at a 400-450 relative centrifugal force for 5 min
3. Pour off the supernatant fluid. Volume of urine and sediment left in the tube should be 0.5 to 1.0 ml
4. Mix the sediment by flicking the end of the tube with the finger, then place a drop (20 ul or 0.02 ml) on a
microscope slide and cover with a cover glass (22x22 mm)
5. Examine with the low power objective to obtain an overall picture of the deposit. Use the HPO to examine
objects more closely.
Manner of reporting (old method of reporting)
a. RBC and WBC - average cells/HPF
- TNTC (too numerous to count)
b. Casts - average casts/LPF
c. Epithelial cells - +, ++, +++, ++++
- Maybe reported as rare, few, moderate, abundant or plenty
d. Crystals - +, ++, +++, ++++
- Maybe reported as rare, few, moderate, abundant or plenty
e. Yeast, bacteria, trichomonas- few, moderate, plenty/ hpf
f. Sperm cells - report in male only
Reporting of crystals and epithelial cells
+ Or rare 1 for every 5 fields
++ Or few 2-5/ field
+++ Or moderate 6-10/field
++++ Or plenty more than 10/field

Microscopic quantitation: 10 fields (new method)

Epith cells/LPF Crystals/HPF
None: 0 None: 0
Rare: 0-5 rare: 0-2
Few: 5-20 few: 2-5
Moderate 20-100 Moderate 5-20
Many >100 Many >20

Bacteria /HPF Mucus thread/ LPF

None 0 Rare: 0-1
Rare 0-10 Few 1-3
Moderate 50-200 Moderate 0-10
Many >200 Many >10

Formed elements Field reporting

Casts /LPF 0, 0-2, 2-5, 5-10, >10
RBCs/ WBCs /HPF 0, 0-2, 2-5, 5-10, 10-25, 25-50, 50-100, >100
 Activity #5
PREGNANCY TESTING

OBJECTIVES: At the end of this activity, the student shall:

1. State the principle of the different pregnancy tests
2. Correlate test results
3. Identify sources of errors and corrective measures
4. Apply concepts of quality assurance and quality control
5. Report test results using the standard format

Methods:
1. Monoclonal Beta- HCG pregnancy test (Indirect)
- A rapid latex slide test for the qualitative detection of human chorionic gonadotrophin (HCG) in urine

Principle:
When a urine sample containing 0.5 IU/ml HCG is mixed with the antibody it effectively binds the antibody on the particles,
thus inhibiting their agglutination (positive result). When a urine sample containing no HCG or less than 0.5 IU/ml is mixed
with the antibody, it is free to react with HCG on the latex causing a visible agglutination (negative result).
Procedure:
1. Bring reagents and specimen to room temperature
2. Place one drop of the HCG positive control on field #1 of the reaction slide. Place one drop (50 uL) of the HCG
negative control on field #2. Use the remaining fields for test specimens
3. Place 1 drop of the specimen on the field
4. Add 1 drop of Beta HCG antiserum to each test field. Mix using stirrer stick
5. Rotate the slide in a circular motion for 30 sec
6. Add 1 drop (50uL) of the HCG latex reagent to each test field. Mix with stirrer stick, spreading reaction mixture over
entire test field
7. Rotate the slide for two minutes and read immediately under direct light.

Positive reaction: uniform, milky suspension with no agglutination within 2 minutes

Negative reaction: observable agglutination in the reaction mixture within 2 minutes

2. Rapid ACU slide test

–a latex agglutination-inhibition test

Principle: when the antibody is mixed with HCG in urine from pregnant women, the antibody is blocked and will not
agglutinate the latex. This is a positive test for pregnancy. Since there is no HCG in the urine of non-pregnant women,
the antibody will not be blocked and the latex will agglutinate. Thus, agglutination of the latex indicates a negative
test for pregnancy
Procedure:
a. Place one drop of urine onto a circle of the slide
b. Add one drop of anti-HCG antibody ( blue) directly on the urine
 c. Add one drop of well shaken HCG –bound latex suspension (yellow)
d. Stir the mixture with the stirrer until it is spread over the entire circle
e. Rock the slide gently for 2 minutes and observe for agglutination
Interpretation of results
Negative: agglutination will occur within 2 minutes
Positive: no agglutination will occur within 2 minutes
3. One step cassette style hCG Urine Pregnancy test
-used to obtain visual, qualitative result for early detection of pregnancy
Principle: Enzyme Immunoassay
Procedure:
a. Open the sealed pouch by tearing along the notch
b. Draw 0.2 ml (about 4 drops ) sample unto the pipette and dispense it into the sample well on the cassette
c. Wait for the colored bands to appear. Positive results may be observed in as short as 40 sec. To confirm
negative results, the complete reaction time (5 minutes) is required. Do not read results after 10 minutes

Interpretation of Results:
Negative: Only one color band appears on the control region. No band on the test region
Positive: distinct color bands appear on the control and test regions
Invalid: No visible band at all. Repeat test with a new test kit.
 Activity #6
STOOL EXAMINATION/ FECALYSIS

OBJECTIVES: At the end of this activity, the student shall:

1. Explain the significance of stool analysis/fecalysis
2. Discuss methods of collection, transport, handling, processing, preservation and proper disposal
3. Characterize an acceptable specimen for routine testing
4. Describe a normal stool
5. Perform the following parameters in a routine stool analysis:
a. Physical examination
b. Chemical examination
c. Microscopic examination
 Activity #5
SEMINAL FUID EXAMINATION

OBJECTIVES: At the end of this activity, the student shall:

1. Explain the significance of seminal fluid analysis
2. Discuss methods of collection, transport, handling, processing, preservation and proper disposal
3. Characterize an acceptable specimen for routine testing
4. Describe a normal seminal fluid
5. Perform the following parameters in a routine semen analysis:
a. Physical examination ( volume, color, pH, liquefaction time, viscosity)
b. Sperm motility
c. Sperm count
d. Sperm morphology
6. Arrive accurate calculation of sperm concentration and sperm count
7. Discuss principle involved in each test performed
8. Correlate test results with pathologic conditions
9. Discuss variables that affect test results
10. Apply concepts of quality assurance and quality control
11. Report test results based on the standard format

Materials:
Per group: per class:
Hemocytometer & tally counter safranin stain
Cold water petroleum jelly
2 slides & 1 cover slip
Dropper, applicator stick
Macroscopic Examination:
Take note of the appearance, volume, pH, viscosity of the semen and the liquefaction time.

Microscopic examination:
1. Motility
a. Hanging drop Method
1. Place 1 drop of semen on a pre-warmed slide
2. Cover with coverslip which has been ringed with petroleum jelly
3. Scan several fields until a total of at least 200 sperm cells have been observed
4. Take note of the percentage of sperm cells showing actual progressive motion
Sperm Motility Grading
4.0 a Rapid, straight line motility
3.0 b slower speed, some lateral movement
2.0 b slow forward progression, noticeable lateral movement
1.0 c No forward progression
0 d No movement
Normal = > 50% motile sperm cells w/in 1 hr.

2. Morphology
Procedure:
1. Prepare a smear (same as in preparation of blood films)
2. Dip once into either 95% ethanol or 50% ethanol ether
3. Air dry
4. Stain with safranin (3-5 dips)
5. Rinse with water
6. Air dry

Examine at least 200 cells under oil immersion objective and take note the percentage of normal and abnormal forms. Observe
appearance of both head and tail.

Normal sperm morphology

Head oval shaped 0.5 um long and 3 um wide acrosomal cap ½ of the head for ovum penetration
Middle piece contains mitochondria- provides energy for flagellar tail motion
Tail 45 um long flagellar tail
Abnormal head double, tapered, giant, amorphous, pinhead, constricted
Abnormal tail double, coiled, bent

3. Sperm cell count and sperm concentration

Normal value - sperm concentration: >20million/ml
Sperm count: >40 million/ejaculation
Diluting fluids- Na bicarbonate, formalin, cold water

Procedure:
a. Suck liquefied semen up to 0.5 mark of WBC pipet
b. Suck diluting fluid up to 11 mark
c. Mix, discard 3-5 drops and charge in a counting chamber
d. Stand for 2 minutes ( for the immobilized sperm to settle)
e. Count sperm cells in 2 sq. mm (2 WBC squares)
Formula: Sperm cells/ml= sperm cells counted x 100,000