Urine specimens are submitted for culture from patients with symptoms of urinary tract infections and from asymptomatic patients with a high risk of infection. The most common gram-negative bacteria found in a urine culture are: Escherichia coli, Enterobacteriaceae sp. Klebsiella spp., Proteus spp., and Staphylococcus saprophyticus.
Quantitative cultures of urine specimens are critical for diagnosis of UTI. The criteria to be used for distinguishing significant from non-significant growth may vary depending on the patient and type of urine specimen received for culture.
MATERIALS AND EQUIPMENT
Sterile urine collection cups
Platinum or plastic disposable calibrated loops (0.001-ml for colony counts>1,000 CFU/ml)
Gram stain reagents
Reagents specified in preliminary identification protocol
1. Specimen collection – Urine is normally a sterile body fluid. However, unless it is collected properly, it can become contaminated with normal flora from the urethra, vagina, prostate or perineum. Laboratory staff must provide detailed instructions to the patients to ensure proper specimen collection.
Clean-catch midstream urine or catheter urine. See Specimen Collection section of this manual for more complete instructions.
2. Timing and transport
a. First morning specimen is best. Excessive fluid intake will dilute the colony count to <105>
3. If the intent of the requisition is to establish whether the infection is Gram-positive or Gram
negative, Grams stain the sediment of urine that has been centrifuged at 1500 g for 5 minutes. Evaluate the organisms OIF. Report whether the organisms are Gram-positive or Gram-negative.
INOCULATION AND INCUBATION
SURFACE STREAK METHOD
The standard choice of media used for initial inoculation of urine is: BA (non-selective media), and MAC. The proper procedure for inoculating urine specimens with a calibrated loop onto culture medium is as follows:
1. Flame and cool a platinum loop calibrated to deliver 0.001 ml (alternatively, a commercially purchased calibrated disposable plastic loop delivering 0.001 ml may be used.)
2. After mixing the urine, remove the top of the container.
3. Holding the cooled loop vertically, immerse the loop below the surface of the urine specimen and subsequently remove the loop vertically.
4. Inoculate the urine on the agar by making a single line steak down the middle of the plate from top to bottom.
5. Beginning at the top of the plate, streak the urine back and forth across the inoculum line, filling the plate with streak lines.
6. Then streak the plate at right angles to the initial streak to produce isolated colonies.
7. Incubate both culture plates at 35 to 37o C in an aerobic atmosphere for 18-24 h.
EXAMINATION OF CULTURE MEDIA
1. Examine cultures that have been incubated overnight.
2. If there is no visible growth and the specimen was collected by voiding or with a catheter, report as follows: “No growth at 24 hours”
3. Re-incubate culture plates with tiny or scant colonies that are not discernible.
4. For positive cultures, examine culture plates for the quantity and morphological type of organisms present. The BA plate is used for the total bacterial count. MAC is inhibitory and could give erroneous quantitative results. MAC should only be used for the presumptive identification of gram-negative rods. A total colony count is performed and the CFU/mL of urine is calculated. Using a 0.001-ml loop, one colony equals 1,000 or 103 CFU/mL (10 colonies would be equal to 10,000 or 104 CFU/ml).
5. Perform additional testing based on the colony count and morphology, method of urine collection, and clinical condition. In routine “clean catch” urine specimens colony counts of 100,000 or 105 CFU/mL or greater are considered significant. Counts of 103 – 105 CFU/mL may also be considered significant from patients on antimicrobial agents, female patients with urethritis, symptomatic males, and dilute urines. Consult with the medical provider as required.
6. In general when there are three or more colony types of organisms grown, regardless of the colony count, it indicates that the specimen was contaminated during collection. This is especially true if the organisms are common vaginal flora such as Lactobacillus sp. Other common contaminants include diphtheroids, viridans streptococci, and coagulase negative staphylococci other than Staphylococcus saprophyticus.
Colony counts should be correlated with clinical criteria to confirm a diagnosis of urinary tract infection (UTI).
1. No Growth
Final report: “No growth at 48 hours”.
2. Colony count <>
a. If mixed:
Final report: “Multiple microorganisms present: probable contamination; please repeat culture”.
b. If pure culture of one or two potential pathogens equally predominant
· Perform definitive identification and antimicrobial susceptibility testing (AST), if indicated. (Refer to Antimicrobial Sensitivity SOP).
· If one organism is predominant and the other is “few”, work up only the predominant organism).
Final report: Enumeration CFU/ml and report organism ID and AST results for each organism.
1. Bacteria in urine may settle to the bottom of the specimen container. Therefore, mixing the specimen prior to sampling is extremely important.
2. A quick visual estimation of the number of colonies of each organism is sufficient.
3. If there is a question on which guideline applies to a given culture result, choose the guideline involving more identification and sensitivity testing.
4. The guidelines for working up cultures are only approximations. They must be modified to fit unusual clinical situations.
5. Do not perform AST directly from the urine specimen.
1. For any urine cultures that grow a significant Stahyococcus coagulase negative, Staph. saprophyticus should be ruled out. It is a very important pathogen among young sexually active females. Staph. Saprophyticus is novobiocin resistant by Kirby-Bauer testing using a disc of 5 ug (<16>