Urinalysis and Acid/Base Balance
Urinalysis and Acid/Base Balance
I. PURPOSE
This exercise is designed to familiarize the student with some of the physiological factors that are involved in kidney function.
II. PERFORMANCE OBJECTIVES
At the end of this exercise the student should be able to:
1. Describe the effects that drinking large volumes of water has on urine production.
2. Describe the effects that caffeine has on urine production.
3. Describe the normal color, odor and pH of urine.
4. Describe the main functions of the kidneys.
5. Describe the physical characteristics of normal urine.
6. Describe the role the kidneys have in the regulation of pH.
III. INTRODUCTION
The kidneys are the most important organs responsible for homeostatic control of blood and other body fluids. They move water and a variety of other substances out of the blood into the environment and in so doing establish a balance between removal and retention of essential and harmful materials. To this end, they help maintain osmotic pressure, electrolyte balance, pH and minimize nitrogenous wastes produced as a result of protein metabolism.
The human kidneys contain more than two million nephrons, the functional units of the kidney, that are responsible for the performance of these functions. Glomerular filtration, tubular reabsorption, and tubular secretion are involved in this process of urine formation. As the filtrate passes through the tubule segments of the nephron, the fluid is modified both in volume and composition as a result of these processes.
Water reabsorption is regulated by antidiurectic hormone (ADH) which is released from the posterior pituitary. Changes in the amount of fluid intake causes osmoreceptors to respond which results in a change in the production of ADH depending on the amount of fluid intake. The ADH has an effect upon kidney tubules to change urine output. For example, an increase in osmotic pressure brought about by a decrease in fluid intake stimulates the production of ADH with resultant retention of fluid and decreases in urine output.
Sodium and potassium regulation is taken care of by the kidneys through the secretion of aldosterone produced by the adrenal glands. Sodium passes in and out of the body and is maintained in the blood at constant levels. If the level becomes high, the kidney can excrete excess sodium. Regulation of sodium is closely related to fluid volume since retention of increased amounts of sodium will result in the retention of additional water. With increasing aldosterone, increased sodium, chloride, and water are retained.
To maintain acid-base balance, the kidneys can eliminate more hydrogen ions if the pH of the extracellular fluid goes down. If the pH goes up, the kidney will eliminate more bicarbonate ions to maintain this proper balance. Even with changing acidity or alkalinity of intake loads, the pH of the blood will remain 7.4 but urine pH will change to maintain this value.
In this experiment, some of the physiological principles mentioned will be demonstrated using your own kidneys as the subject for this investigation.
Part I (all students except for those participating in part II)
Safety Precautions:
In this lab you will be using your own urine as one of the specimens that you will be analyzing. You must follow the usual safety precautions for working with body fluids:
1. Wear gloves for the entire lab
2. For student specimens, test and handle only your own urine
3. Follow proper disposal procedures as described below
4. After you finish the lab, make sure all supplies are returned to their proper places and wipe
down your lab bench with disinfectant
Activity 1: Analyzing Your Own Urine Sample
Use the materials available to analyze your own urine sample.
Record your results in the table on your data sheet.
For physical characteristics: use terms from the table below to describe urine color & odor:
| |Normal Values |Abnormal Values |
| |Colorless |Milky |
|Color |Pale Straw |Reddish Amber |
| |Straw |Brownish Yellow |
| |Amber |Green |
| | |Smoky Brown |
|Odor |Aromatic |Maple Syrupy |
| |Musty |Sweet |
| |Ammonia-like (old) |Malt |
| | |Acetone-like |
*Do not analyze for inorganic components
*Use the urine test strip and pH paper to determine pH of your urine sample;
DO NOT USE THE PH METER
Activity 2: Analyzing ‘Unknown’ Urine Samples
Analyze each of the unknown urine samples by using the urine test strips (dipsticks), record the results in the table below; Do not use the urinometers or pH meters to analyze the unknown urine samples then suggest a diagnosis that might explain any of the abnormal results that you obtained.
*Place an asterisk next to any “abnormal” values in these samples
Activity 3: Analyzing Urine Sediment Microscopically
1. Place 10 ml of urine in the centrifuge tube and screw on cap; centrifuge for 5 minutes
2. The instructor will centrifuge your urine sample
3. Prepare a slide:
a. pour out all but the last ml of urine from the centrifuge tube
b. add a drop of sedi stain to the centrifuge tube, replace the lid and mix use a disposable pipette to collect ONE drop of stained urine from the centrifuge tube and place this drop on a clean slide
4. Place a coverslip over the drop on the slide
5. Draw and attempt to identify some of the different kinds of sediment in your sample. Use illustrations in the handout provided
II. Acid /Base Balance
Activity 4: Determining the pH of biological solutions using pH paper
1. Take a small amount of each of the following solutions and use the pH paper provided to determine their pH:
a. Saliva
b. Urine (take average value of pH from Urinalysis lab
c. Plasma (sample provided)
2. Record your results in the table on your data sheet
Activity 5: Effects of buffers on acidic and alkaline solutions:
a. Hydrochloric Acid (HCl) in deionized water
1. Rinse all glassware with deionized water
2. Fill a 125ml Flask to the 50 ml mark with deionized water
3. Use the pH meter to record the initial pH of the solution by immersing the electrode and stirring
it in the solution
4. Record the pH on the table on your data sheet.
5. Immediately add HCl solution drop by drop while stirring with the pH electrode continuously.
Continue to slowly add and count the drops of HCl until the pH changes by one complete unit.
6. Record the final pH of the solution and the number of drops of HCl in the table on your data sheet.
7. Remove the pH electrodes and rinse them with deionized water
b. Sodium Hydroxide (NaOH) in deionized water
1. Repeat the procedure a above, but this time adding the NaOH solution drop by drop and record
your results in the table on your data sheet.
c. HCl in Sodium Carbonate
1. Fill another 125ml Flask to the 50 ml mark with 0.05M NaHCO3 (sodium carbonate) solution
2. Use the pH meter to record the initial pH of the solution by immersing the electrode and stirring
it in the solution.
3. Add HCl drop by drop, counting the drops while stirring with the pH electrode, until the pH
changes one complete unit.
4. Record the final pH of the sodium carbonate and the number of drops of HCl used in the table on your data sheet.
5. Remove and rinse the electrode with deionized water.
d. NaOH in Sodium Carbonate
1. Repeat the procedure c above, but this time adding the NaOH solution drop by drop and record
your results in the table on your data sheet.
e. HCl in Plasma
1. Place 50 ml of dilute plasma into a clean 150 ml flask, and record its initial pH with the pH meter. Ask your instructor what the dilution factor is.
2. Add HCl drop by drop, counting the drops while continuously stirring with the pH electrode, until the pH changes one complete unit.
3. Record the final pH of the plasma and the number of drops of HCl used in the table on your data
sheet.
4. Remove and rinse the electrode with deionized water.
f. NaOH Plasma
1. Repeat procedure e above this time using the NaOH solution and record your results in the table on your data sheet.
Part II (Student Volunteers)
V. PROCEDURE
Food and water intake should be restricted for approximately two hours before starting this exercise. Persons with kidney abnormalities or problems should be eliminated from the ingestion of test solutions but should aid their group members in the analysis of urine samples.
1. Before drinking any of the test solutions each student should urinate and keep this sample for further analysis.
2. The students should work in groups of 3 or 4 depending on class size. All students in the group should be drinking the same experimental solution.
3. Students should drink all solutions quickly but without discomfort and should be divided up into the following groups:
Group A - drinks 800-1000 ml of water
Group B - drinks 300 ml of a 2.5% sodium bicarbonate
Group C - drinks 800-1000 ml of coffee or tea (without sugar)
4. Each student should urinate at 30 minute intervals , collect the urine and measure its pH, specific gravity, sodium chloride content, and glucosecontent using methods described in this exercise. If you are unable to void, a urine sample is taken at the next 30 minute interval.
5. Your urine sample from step 1 and those taken at 30 minute intervals shouldbe evaluated using the following procedures for urinalysis. All data should be recorded in the chart at the end of this laboratory exercise.
Color - Normal urine is straw to amber color. Observe the color of urine before ingestion of test solutions and note any changes that occur during the course of this experiment.
Volume - Measure the volume of the urine specimen by pouring it into a graduated cylinder.
Specific Gravity - The normal range for specific gravity is between 1.015 and 1.030. It is a measure of the relative amounts of solids in the urine. If possible, use a refractometer.
[pic]
Directions for Refractometer
1. Calibrate the refractometer by placing distilled water on the glass as the sample, and adjusting the scale to read 1.000.
2. Open up the flap at the end of the refractometer. Clean by rinsing with distilled water while the end of the refractometer is held over a large beaker labeled "Urine Waste" . Dry the glass tip of the refractometer with a Kimwipe. Place a drop of urine on the glass plate and gently close the flap. Hold the refractometer up towards an area of natural light, look though the eye piece and read the specific gravity level off the scale - the point where the contrast line (difference between light and dark areas) crosses the scale. After each use, clean with distilled water and dry with a Kimwipe as described above. The Kimwipes should then be placed in one of the biohazard bags. The presence of glucose and some medicines may cause the urine specific gravity to change and give incorrect readings. If any of these situations are possible, the glass urinometer will have to be used.
Directions for Urinometer (if refractometer unavailable)
To measure specific gravity, place the hydrometer (urinometer) in its cylinder filled 3/4 full of urine. Remove any bubbles on the surface of the urine by rotating the urinometer. Also make sure that the urinometer does not cling to the sides of the urinometer cylinder. Observe where the urine meniscus lines up with the lines engraved on the urinometer for the value of specific gravity. Specific gravity changes with temperature and should be corrected. Add .001 for every 3o C above 15o C. Use the same urinometer for all tests during the course of the experiment and make sure that it is washed, rinsed and dried after each use.
Glucose-
Use the test strips available to determine glucose
pH
Record the pH using a pH meter and follow procedures that your instructor will provide for sample testing . Clean the probe each time you take a reading. To clean the probe, hold the probe over one of the beakers labeled "Urine Waste" and using the squeeze bottle filled with distilled water, squeeze gently and produce a light stream of water to clean the probe. Blot the probe dry with a Kimwipe. The Kimwipes should be discarded in the trash.
[pic]
Sodium Chloride Determination
a. Measure 10 drops of urine into a small test tube
b. Add 1 drop of 10% potassium chromate
c. Add 2.9% silver nitrate 1 drop at a time and shake after each drop
d. Count the number of drops required to turn the solution from yellow to brown
e. Calculate the sodium chloride content as follows:
Na Cl (gm) = volume of specimen in ml 1 x drops silver nitrate
1000 ml
Proteins, ketones, blood, bilirubin
With your urine and a pathological urine if available, use multistix to determine protein, ketones, blood, and bilirubin.
Microscopic Examination
1. Obtain 10 ml of freshly voided urine from the first voided control sample.
2. Centrifuge for 10 minutes at 1600 rpm.
3. Place one drop of sediment on a clean glass slide.
4. Add one drop of sedi-stain and place a cover glass over specimen.
5. Under low power look for the presence of blood cells, salt crystals, epithelial cells, bacteria, yeast cells, parasites and casts (Figure 1).
6. Under high power (40x) count five fields and average the number of red and white cells found.
7. Enter your observations and data in the chart at the end of this laboratory exercise.
Disposal: slides, coverslips( glass disposal box
gloves, towels, cups, urine test strips, straws ( trash
MATERIALS (for part II)
2.5% sodium bicarbonate, 50% sucrose solution (Glucola) caffeinated coffee, tea, hot water, urine specimen containers, pH paper and pH meter, buffer (pH7), 500 ml graduated cylinders (100ml and 500 ml, urinometers, refractometers, thermometers, 10% potassium chromate, 2.9% silver nitrate, multistix, large test tubes, small test tubes, centrifuge tubes, 10 ml pipets, pipeting devices, test tube racks, test tube holders, microscopes, sedi-stain, cover slip, rubber gloves, centrifuge.
[pic]
Urinalysis and Acid/Base Balance
Data Sheet Part I
I. Urinalysis
Activity: Your Own Urine Sample
|Urinalysis Results |
|Physical Characteristics | |
| |Color | |
| |Transparency | |
| |Odor | |
| |pH test paper) | |
| |Specific |“labstick” | |
| |Gravity | | |
| | |refractometer | |
|Organic Components | |
| |Nitrite | |
| |Urobilinogen | |
| |Protein (albumin) | |
| |Blood | |
| |Ketone | |
| |Bilirubin | |
| |Glucose | |
Suggest an explanation for any abnormal components in your urine sample:
Activity: Analyzing Urine Sediment Microscopically
|Sediment Analysis (diagram some examples then try to identify each) |
| |
| |
| |
| |
| |
| |
| |
| |
| |
Describe and try to identify some of the more common types of sediment
Activity: ‘Unknown’ Urine Samples
|Urinalysis Results - Unknowns |
|Physical Characteristics |
| |A |B |C |D |
| |Color | | | | |
| |Transparency | | | | |
| |Odor | | | | |
| |pH | | | | |
| |Specific Gravity | | | | |
|Organic Components (labsticks) |
| |Nitrite | | | | |
| |Urobilinogen | | | | |
| |Protein (albumin) | | | | |
| |Blood | | | | |
| |Ketone | | | | |
| |Bilirubin | | | | |
| |Glucose | | | | |
| | | | | |
|Diagnosis: | | | | |
| | | | | |
| | | | | |
II. Acid /Base Balance
Activity: Determining the pH of biological solutions using pH paper
|Biological Fluid |pH |
|Saliva | |
|Urine | |
|Plasma | |
Which body fluid was the most acidic? Which was the most alkaline? Explain.
Compare your body fluids pH values with those of your class mates. For which of the three body fluids do you think you obtained the most accurate pH reading from? The least accurate? Explain
Activity: Effects of buffers on acidic and alkaline solutions:
|Test |initial pH |final pH |# drops of |# drops |
|Solution | | |HCl |NaOH |
|Water | | | |xxxxxxxxxxxx |
| | | |xxxxxxxxxxx | |
|NaHCO3 | | | |xxxxxxxxxxxx |
| | | |xxxxxxxxxxx | |
|Plasma | | | |xxxxxxxxxxxx |
|dilution factor=____ | | |xxxxxxxxxxx | |
Data Sheet PartII
|General |Normal Urine | |0.5 hr |1.0 hr |1.5 hr |2.0 hr |2.5 hr |3.0 hr |
|Characteristics & | |Sample | | | | | | |
|Tests | | | | | | | | |
|Color |light yellow or | | | | | | | |
| |amber | | | | | | | |
|Odor |pungent | | | | | | | |
| |characteristic | | | | | | | |
|pH |4.5 - 8.0 | | | | | | | |
|Glucose |Neg | | | | | | | |
|Protein |Neg | | | | | | | |
|Volume |1 - 1.5 L/day | | | | | | | |
|Specific Gravity |1.001 - 1.030 | | | | | | | |
|Sodium Chloride |present | | | | | | | |
|Bilirubin | | | | | | | | |
|Ketones |neg | | | | | | | |
|Sediment | | | | | | | | |
|WBC's |neg | | | | | | | |
|RBC's |neg | | | | | | | |
|Epithelial Cells |present | | | | | | | |
|Casts |neg | | | | | | | |
|Crystals |neg | | | | | | | |
|Bacteria |neg | | | | | | | |
|Parasites |neg | | | | | | | |
|Yeast Cells |neg | | | | | | | |
Review Questions:
1. If the specific gravity of the urine specimen is high, what color would you expect the sample to be? Explain.
2. What specific conditions would result in urine of high specific gravity? Of low specific gravity?
3. List five examples of abnormal constituents in a urine sample (other than those you found in your unknown samples above) and describe what problems each might indicate
4. How do you account for the fact that saliva and urine may have a pH below 6.8 or above 7.8 in healthy individuals.
5. What exactly is a buffer? Which of the three solutions (water, bicarbonate, plasma) acted as the least effective buffer? Explain.
6. Compare the effects of acids and bases on the buffered and unbuffered solutions. How does the buffering ability of plasma or albumin compare with that of sodium bicarbonate?
1 Amount of urine produced at each test period
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