Quantitative Urine Culture

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 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).
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.

Purpose	To learn quantitative culture technique and to see the effects of delay in culturing a voided urine specimen
Materials	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

Procedures

1. 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.
2. Incubate the plate at 35°C for 24 hours.
3. 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.
4. Read your plates, count the colonies on each, and report the numbers of organisms per milliliter present in the urine specimen.
5. Inspect the tubes of urine left in the refrigerator, in the incubator, and on your bench. Examine for turbidity and record results.

Results

1. Record the number of colonies on the streaked nutrient agar plate.
2. Calculate and record the number of organisms per milliliter of specimen and indicate whether this result is significant of urinary tract infection.
3. Diagram your observations of turbidity in each tube of stored urine.
   What is your interpretation of the appearance of these tubes?