To distinguish contamination of urine by normal urogenital flora from urinary tract infection 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
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
In this experiment, you will have a simulated urine specimen from a suspected case of urinary tract infection submitted
with a request for quantitative culture.
||To learn quantitative culture technique and to see the effects of delay in culturing a voided urine
||Nutrient agar plates
Calibrated loop (0.01-ml delivery)
Sterile 5-ml pipettes
Pipette bulb or other aspiration device
Sterile empty tubes
Simulated “clean-catch” urine from a clinical case of urinary tract infection
- With the calibrated loop, transfer 0.01 ml of the urine specimen to the center of a nutrient agar plate and streak across the
drop in several planes so that the specimen is distributed evenly across the plate.
- Incubate the plate at 35°C for 24 hours.
- Go back to the original urine specimen and measure about 2.0 ml into each of two sterile, empty test tubes. Place one of
these in the refrigerator, leave one at room temperature at your station, and place the original specimen in the incubator
for 24 hours.
- Read your plates, count the colonies on each, and report the numbers of organisms per milliliter present in the urine
- Inspect the tubes of urine left in the refrigerator, in the incubator, and on your bench. Examine for turbidity and record
- Record the number of colonies on the streaked nutrient agar plate.
- Calculate and record the number of organisms per milliliter of specimen and indicate whether this result is significant of
urinary tract infection.
- Diagram your observations of turbidity in each tube of stored urine.
What is your interpretation of the appearance of these tubes?