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After completing this exercise, students should be able to:
1. Describe three ways in which urinary tract infections may be acquired.

2. Explain the difference between significant and insignificant bacteriuria.
3. Define cystitis, pyelonephritis, and acute urethral syndrome.
4. Name three gram-negative and three gram-positive bacteria that may cause urinary tract
infections.
5. Instruct male and female patients on the proper collection of a clean-catch, midstream urine
sample.
Normally, urine is sterile when excreted by the kidneys and stored in the urinary bladder. In health, the
only portion of the urinary tract that contains a normal flora is the terminal third of the urethra. When
urine is voided, however, it becomes contaminated by the normal flora of the urethra and other superficial
microorganisms found on urogenital mucous membranes. The presence of bacteria in voided
urine (bacteriuria), therefore, does not always indicate urinary tract infection. To confirm infection,
either the numbers of organisms present or the species isolated must be shown to be significant.
Active infection of the urinary tract develops in one of three ways: (1) microorganisms
circulating in the bloodstream from another site of infection are deposited and multiply in the
kidneys to produce pyelonephritis by the hematogenous (originating from the blood) route;
(2) bacteria colonizing the terminal third of the urethra or the external urogenital surfaces ascend
the urethra to the bladder, causing cystitis (infection of the bladder only) or pyelonephritis by the
ascending
route; or (3) microorganisms, usually from the urethra, are introduced into the bladder
on catheters or cystoscopes.
Cystitis is much more common than pyelonephritis. In the former case, most of the
offending
organisms are opportunistic members of the fecal flora, including many of the gramnegative
bacteria you have studied in Exercises 23 and 24, such as Escherichia coli (by far the
most frequent cause of urinary tract infection). Klebsiella, Enterobacter, Serratia, and Proteus,
Pseudomonas, and Acinetobacter are also incriminated, especially in hospitalized patients with
indwelling
urinary catheters or those receiving multiple antimicrobial agents. When these organisms
reach the bladder, where active host-defense mechanisms (blood phagocytes and antibodies)
are not readily available, they may grow in the urine, producing acute bladder infection. Among
the gram-positive bacteria, Enterococcus spp., Staphylococcus saprophyticus and other coagulasenegative
staphylococci, and Staphylococcus aureus may also cause urinary tract infections.
The blood that flows through the kidneys normally carries no microorganisms because
phagocytic white blood cells and serum antibodies are constantly at work eliminating any microbial
intruders that reach deep tissues. If these defense mechanisms are not working well or become overwhelmed
by extensive infectious processes in systemic tissues (uncontrolled tuberculosis or yeast
infections, staphylococcal or streptococcal abscesses), then the kidneys may become infected by organisms
carried to them via the bloodstream. More commonly, however, pyelonephritis results from
microorganisms initially infecting the bladder and then ascending the ureters to infect the kidneys.
Laboratory diagnosis of urinary tract infection is made by culturing urine, usually obtained
either by catheterization or by voided collection. To obtain a catheterized urine specimen,
a sterile, polyurethane catheter tube is inserted into the urethra and passed up into the bladder. The called “acute
urethral syndrome,” is thought to occur in a subset of patients, usually women, who
have symptoms of urinary tract infection. These patients require treatment, even when their urine
cultures show low colony counts or no growth of bacteria. Close collaboration between the laboratory
and the physician is needed to accurately diagnose these infections.
Collection of Voided Urine for Culture
(“Clean-Catch” or “Clean-Voided” Techniques)
Aseptic urine collection requires careful cleansing of the external urogenital surfaces, using
gauze sponges moistened with tap water and liquid soap. Special “clean-catch” urine collection
kits are also available in many medical facilities. They contain moistened towelettes for proper
cleansing of the genitalia and a sterile cup with a leak-proof, screw-cap lid for urine collection.
For males, the procedure simply entails thorough washing of the glans of the penis,
discard of the first stream of urine, and collection of a “midstream” portion in a sterile container.
If the outside of the container has been soiled with urine in the process, it must be wiped clean
with disinfectant before being handled further.
For females, extra care is necessary. All labial surfaces must be thoroughly cleansed,
and the sterile container must be held in such a way that it does not come in contact with the skin
or clothing. Again, the first stream of urine is discarded, and a midstream sample is collected.
When the container has been tightly closed, it is wiped clean with disinfectant. The proper
procedures
for the collection of “clean-voided, midstream” urine samples from male and female
patients are shown in figure 25.1.
Figure 25.1 Procedure for collecting a clean-voided midstream urine sample.
1. Wash your hands
with soap and water.
Women: Wipe between
the folds of your genitals
from front to back.
Men: Wipe the head of your
penis and the opening.
(Uncircumcised men pull
foreskin back.)
3. Wash your genitals using packaged towelettes or cotton balls with soap and water.
6. Put the lid tightly on the cup and return it as directed.
Be certain it is labeled appropriately.
2. Remove the lid from the
cup. Place it face up on
the counter. Do not touch
the inside of the cup or lid.
4. Start to urinate into the toilet. Then,
during midstream, move the cup into the
stream to catch some urine. This will
ensure that there are no bacteria in the
urine sample.
5. Pull the cup away before you finish
urinating when the cup is about half full.
Do
Urine containers should never be filled to the brim. Closures should be doublechecked
to make certain they will not permit leakage during transport to the laboratory. If there
is any delay (before or after delivery to the lab) in initiating culture, urine specimens must be
refrigerated.
EXPERIMENT 25.1 Examination and Qualitative Culture of Voided Urine
Purpose To learn simple urine culture technique and to appreciate the value of clean-voided urine
collection
Materials Urine sample no. 1
Urine sample no. 2
Sterile empty test tubes
Sterile 5.0-ml pipettes
Pipette bulb or other aspiration device
Litmus or pH papers
Blood agar plates
MacConkey plates
Marking pen or pencil
Test tube rack
Procedures
1. Your instructor will provide you with two simulated urine samples labeled numbers 1 and 2. Sample number
1 represents a
urine sample collected without any special cleansing of the urogenital surfaces, whereas sample number 2 was
collected
using the appropriate “clean-voided” technique.
2. Place about 1.0 ml of each urine sample in small sterile test tubes labeled “no. 1” and “no. 2.” Hold the tubes
to the light and
examine urine for color and turbidity. Test the pH of each sample with litmus or pH paper. Record your
observations under
Results.
3. Going back to the original urine container (the test tube sample is now contaminated by the pH test), pipette a
large drop
of the “clean-voided” specimen (no. 2) onto a blood agar plate near the edge, and another drop onto a
MacConkey plate.
Spread the drop a little with your loop and then streak for isolation. Label the plates “no. 2.”
4. Repeat step 3 with the casual urine collection (no. 1), labeling the plates “no. 1.”
5. Incubate all plates at 35°C for 24 hours.
6. Examine the incubated plates for amount of growth, types of colonies, and microscopic morphology of colony
types.
Record
observations under Results.
Results
1. Macroscopic
EXPERIMENT 25.2 Quantitative Urine Culture

To distinguish contamination of urine by normal urogenital flora from urinary tract infection caused by the same
organisms, it
is usually necessary to determine the numbers of organisms present per milliliter of specimen. In general, counts
in excess of
100,000 organisms per milliliter are considered to indicate significant bacteriuria, if the collection technique
was adequate and
there was no delay in culturing the specimen.
A quantitative culture is prepared by placing a measured volume of urine on an agar plate and counting the
number
of colonies that develop after incubation. A calibrated loop that delivers 0.01 ml of sample is used to inoculate
the plate. The
number of colonies that appear from this 1/100th-ml sample is multiplied by 100 to give the number per
milliliter. For example,
if 15 colonies are obtained from 0.01 ml, there are 15 × 100, or 1,500, organisms present in 1 ml (assuming each
colony represents
one organism). Some clinical microbiology laboratories use a 0.001-ml calibrated loop instead of a 0.01-ml loop
for
performing quantitative urine cultures. In this case, the number of colonies that grow is multiplied by 1,000 to
give the total
colony count per milliliter. For example, if 15 colonies are obtained from 0.001 ml of urine sample, there are 15
× 1,000 or
15,000 colonies per milliliter of urine.

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After completing this exercise, students should be able to:

1. Describe three ways in which urinary tract infections may be acquired.

EXPERIMENT 25.2 Quantitative Urine Culture

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