1. Lab 14: Urinary System (Part II)
Dr. Kim Wilson

2. INTRODUCTION
The analysis of urine has 2 purposes. One is to detect body disturbances, such as endocrine or metabolic abnormalities, in which the kidneys function normally but excrete abnormal amounts of metabolic end products specific for a particular disease. The second purpose is to detect disease conditions within the kidneys or urinary tract themselves that may adversely affect the urinary system's ability to function efficiently in the maintenance of fluid, electrolyte & acid‑base balance. Urine may be analyzed both physically & chemically using relatively simple tests in order to check for abnormalities. In this lab, a few of these physical & chemical tests will be conducted on a sample of your own urine. For reference, you should review information pertaining to urinalysis in your textbook and in your lab manual (Unit 21).

3. PART 1: APPEARANCE AND PHYSICAL PROPERTIES OF URINE
INSTRUCTIONS:
Obtain a clean specimen cup and collect a sample of your urine. We won't be testing the specimen for the presence of micro‑organisms, however, obtain a "midstream" specimen since it yields the best results.
Pour part of your specimen (approx. 10 ml) into a test tube and cap it. This portion will be allowed to set for 1 hour and then centrifuged in order to microscopically examine its sediments. Use the remaining portion of your specimen to perform the following physical and chemical tests.

4. PART 1: APPEARANCE AND PHYSICAL PROPERTIES OF URINE
A. COLOR
Freshly voided urine is often described as having a straw or amber colored appearance. However, the normal color (which is due to the presence of a pigment known as urochrome) may range considerably from person to person. Concentrated urine, resulting from loss of water in severe sweating, will be dark yellow; a light amber yellow is more characteristic of dilute urine. Certain deviations from the normal amber color may be the result of disease, whereas other color changes may have no pathological significance. For example, a brownish yellow or green color may be the result of bile pigments, suggestive of liver disease. Likewise, a smoky brown or reddish‑brown colored urine usually indicates disease, but may (on occasion) be the result of ingestion of plant dyes found in beets or rhubarb.
Record color on Results Sheet.

5. PART 1: APPEARANCE AND PHYSICAL PROPERTIES OF URINE
B. CLARITY (CLOUDINESS)
A freshly voided urine sample should be clear or transparent. If urine is cloudy when voided or becomes cloudy after standing, the sample should be checked (by microscopic examination & chemical tests) for the presence of mucous, pus, bacteria, sperm, phosphate, or urate crystals.
Record clarity on Results Sheet.
C. ODOR
Normal, freshly voided urine has a "characteristic" odor which is believed to be due to the presence of volatile acids. Urine that has been standing for a long time at room temperature develops an ammonia‑like odor which is due to the decomposition of urea by bacteria in the specimen. The urine of patients with diabetes mellitus may have an acetone odor due to the presence of ketones. The urine of patients with urinary tract infections may be foul‑smelling, especially when the infecting organism is a coliform bacillus. Abnormalities in urine odor may simply be due to the ingestion of certain foods; for instance, garlic and asparagus can give urine characteristic odors.
Record odor on Results Sheet.

6. PART 1: APPEARANCE AND PHYSICAL PROPERTIES OF URINE
D. REACTION (pH)
The pH of urine is a measure of its hydrogen ion concentration. A pH below 7 indicates acid urine; a pH above 7 indicates alkaline urine. Freshly voided urine from persons not on special diets is usually acid and has a pH of approximately 6.0; however, normal kidneys are capable of producing urine that can vary from a pH of 4.5 to a pH of 8. These variations seen in pH may be related to diet, medication, or disease. For example, persons on high protein diets produce a more acid urine whereas persons on diets high in vegetables, milk or other dairy products produce an alkaline urine. Persons with diabetes mellitus (uncontrolled) excrete urine containing large amounts of acid whereas persons with urinary tract infections or persons taking certain antibotics will excrete an alkaline urine.
INSTRUCTIONS:
1. Place a few ml's of fresh urine into a small cup. Dip a strip of pH test paper into the sample 2 or 3 times.
2. Tap the pH paper on the edge of the cup to remove excess urine. Wait one minute & record the pH on the Results Sheet.

7. PART 2: CHEMICAL COMPOSITION OF URINE
A. GLUCOSE
Glucose is the sugar most commonly found in urine, although other sugars such as lactose, fructose, or galactose may also be found under certain conditions. The presence of detectable amounts of glucose in urine is known as glycosuria, and it may either occur in normal or abnormal conditions. For example, glucose may normally spill into the urine following consumption of a heavy meal, especially one that contains large amounts of carbohy‑drates, or in conjunction with emotional stress. The most common abnormal condition resulting in glycosuria is diabetes mellitus. There are several different tests available which check for the presence of glucose in urine.

8. INSTRUCTIONS:
Test # 1: Glucose Determination Using Clinitest Reagent Tablets.
1. Place 10 drops of water & 5 drops of urine in a test tube.
2. Add 1 Clinitest tablet. *Note: Handle Clinitest tablets with extreme caution, using gloves. These tablets contain caustic soda which is highly sensitive to moisture from air or water Excessive moisture may cause chemical burns or bottle explosion.
3. Watch while a complete boiling reaction takes place (Boiling occurs because the concentrated sodium hydroxide in the tablet generates heat.) Do not shake the test tube during the boiling or for the following 15 seconds after the boiling has stopped.
4. After the 15 seconds waiting period, gently shake the test tube to mix the contents. Compare the color in the tube to the standard color chart (for 5 drop method, not 2 drop method).
5. Record results as Negative, Trace, 1+, 2+, 3+, or 4+ on the Results Sheet.

9. Test # 2: Glucose Determination Using Diastix
1. Pour a small amount of urine into a small cup.
2. Dip the reagent end of the strip in urine (do not touch the reagent end with your hands). Remove immediately.
3. While removing, draw the edge of the strip against the rim of the cup to remove excess urine.
4. Compare the reagent side with the corresponding color chart (on bottle) at exactly 30 seconds.
5. Record results on Results sheet.

10. Test # 3: Glucose Determination Using Benedict's Test
1. Place 5 ml of Benedict's solution in a test tube.
2. Add exactly 8 drops of urine and boil the solution for 2 to 5 minutes.
3. If the solution remains blue, the test is negative; green is recorded as +, yellow as ++, orange as +++, and red as ++++.
4. Record results on Results sheet.

11. B. KETONES
Normally the body completely metabolizes fats to carbon dioxide and water. Whenever there is inadequate carbohydrate intake or a defect in carbohydrate metabolism, the body metabolizes increased amounts of fatty acids. When this increase is large, fatty acid utilization is incomplete and intermediate products (Ketones) appear in the blood and are excreted in the urine. Diabetes mellitus is the most important disorder in which ketonuria (presence of ketones in urine) occurs. As with glucose, there are several different tests available which check for the presence of ketones.

12. INSTRUCTIONS: Test # 1: Ketone Determination Using Acetest Reagent
Tablets
1. Place an Acetest tablet on a clean dry piece of paper and put 1 drop of urine on the tablet.
2. At 30 seconds, compare the color formed with the color chart.
The color will remain unchanged or cream‑colored in a negative test, but will vary from pink to purple, depending on the amount of ketone bodies present, in a positive test.
3. Record results on Results Sheet.

13. Test # 2: Ketone Determination Using Ketostix Strips
Place a small amount of urine in a small cup.
Dip the reagent end of the strip in urine (do not touch the reagent end with your hands). Remove immediately.
3. While removing, draw the edge of the strip against the rim of the cup to remove excess urine.
4. Exactly 15 seconds after removing the strip from the specimen, compare the reagent side with the corresponding color chart (on bottle). The color will remain unchanged or appear cream colored (from wetting) if negative; if positive, the color will vary from pink to purple depending upon the amount of ketones present.
5. Record results on Results Sheet.

14. C. PROTEIN
Normally, between 40 and 80 mg of protein is excreted daily, but as much as 100 ‑ 150 mg per day may be considered within normal limits. Typically however, this normal protein isn't detected in the urine. Proteinuria refers to an increased amount of protein in the urine and is one of the most important indicators of renal disease; however, this condition may also intermittently occur in the absence of disease ‑ usually as a result of strenous physical exercise or severe emotional stress. Several tests are available for measuring urinary protein.

15. INSTRUCTIONS: Test #1: Protein Determination Using Albustix Reagent
Strips.
1. Place a small amount of urine in a small cup.
2. Dip the reagent end of the strip in the urine (do not touch the reagent end with your hands). Remove immediately.
3. While removing, draw the edge of the strip against the rim of the cup to remove excess urine.
4. Immediately after removing the strip from the urine, compare the reagent side with the corresponding color chart (on bottle).
5. Record results on Results Sheet.

16. D. BILE SALTS
Bile salts are an essential component of bile. Normally the majority these
substances are recycled in the body with only small amounts lost in urine (the
loss usually isn't detectable). Hay's Test for bile salts involves the addition of
sulfur to urine. Since bile salts lower the surface tension of the water in urine, if
bile salts are present, the sulfur will sink.
INSTRUCTIONS:
Hay's Test for Bile Salts in Urine
1. Place a small amount of urine in a small cup.
2. Sprinkle a little finely powdered sulfur on the surface of the urine. If the sulfur sinks at once, bile salts are present in the amount of 0.01% or more. If the sulfur sinks only after gentle agitation, bile salts are present in the amount of % or more. If the sulfur remains floating even after gentle shaking, bile salts are absent.
3. Record results on Results Sheet.

17. E. COMBINATION TESTS INSTRUCTIONS:
Combistix Method (Determination of glucose, protein, & pH)
1. Place a small amount of urine in a small cup.
2. Dip the reagent end of the Combistix strip in urine ( do not touch the reagent end with your hands). Remove immediately.
3. While removing, draw the edge of the strip against the rim of the cup to remove excess urine.
4. The yellow part of the strip is the indicator for protein. It should be read immediately and compared with the color chart (on bottle).
5. The aqua part of the strip is the test for glucose. This should be read after 30 seconds by comparing the strip to the color chart (on bottle).
6. The orange part of the strip is the pH indicator. It should be read immediately and compared with the color chart (on bottle).
7. Record results on Results sheet.

18. PART 3: THE MICROSCOPIC EXAMINATION OF SEDIMENT OBTAINED FROM URINE
INSTRUCTIONS:
1. Obtain the urine which has set in the test tube for 1 hour.
2. Read the Discussion (below) and complete the Procedure (below).

19. PROCEDURE
1. Place 10 ml of fresh urine in a centrifuge tube and centrifuge for 5 minutes. (Precaution: Make certain the centrifuge is balanced with another tube containing 10 ml of urine or water.)
2. Pour out all the urine possible (do this quickly without shaking the tube or inverting it for more than a moment).
3. Add 2 drops of Sedi‑Stain to the sediment in the tube. Thoroughly mix the contents of the tube with the stain by tapping briskly on the bottom of the tube with the index finger.
4. Transfer 1 drop of the stained sediment onto a clean glass slide.
5. Cover the drop of sediment with a cover glass and examine it under the microscope using both low and high power objectives.
6. Identify as many of the substances in the sediment as possible by comparing them with Figures 1 ‑ 4 (see attached page ‑ this handout), as well as with the urine chart (on the materials table in the lab). Make a drawing of your observations (white paper, high power). Be certain to properly label your drawing.
7. Include your drawing in your lab report.
ANSWER THE QUESTIONS FROM THE QUESTIONS SHEET.