PRINCIPLE
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
1. Media
BA
MAC
2. Supplies
Sterile urine collection cups
Platinum or plastic disposable calibrated loops (0.001-ml for colony counts>1,000 CFU/ml)
3. Reagents
Gram stain reagents
Reagents specified in preliminary identification protocol
API 20E
4. Equipment
Non-CO2 incubator
Microscope
SPECIMEN
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>
b. Transport urine to laboratory as soon as possible after collection.
c. Culture urine specimens within 2 h, or refrigerate and culture within 24 h. Bacterial counts usually remain stable for 24 h at 4oC.
d. Request a repeat specimen when the collection time and method of collection have not been provided.
e. If an improperly collected, transported, or handled specimen cannot be replaced, document in the final report that specimen quality may have been compromised.
QUALITY CONTROL
Inspect calibrated loops regularly to confirm that they remain round and are free of bends, dents, corrosion, or incinerated material. Additionally, at least monthly, check the loops to ensure that the delivery volume is accurate using the drill bit method: Drill bit method: Obtain two twist drill bits (no. 53 or 54). Carefully slip the 0.001-ml loop over the end of the no. 54 bit. If the loop is calibrated, it will fit over the bit. Repeat the procedure with the no. 53 bit. If the loop is calibrated, it will not fit over the end. Discard the loop if it fits over the no. 53 bit. Perform media, reagent, and stain quality control as directed in the Quality Control SOP.
GRAM STAIN (done by request only)
1. The Gram stain method can detect the presence of both bacteria and WBC’s in urine specimens. If an order is received consult with medical provider.
2. If the intent is to determine if there is a high colony count infection, place 0.01 ml of well mixed, uncentrifuged urine onto a glass slide, allow it to air dry without spreading, and Gram stain. (Refer to Gram Stain SOP). Report the number of microorganisms per oil immersion field (OIF). The presence of one or more microorganisms per OIF correlates with a colony count of > 105 CFU/mL. Report and quantitate WBC’s and organisms seen. The presence of many squamous epithelial cells and different microbial morphotypes indicates contamination or “dirty-catch”. Request a repeat specimen.
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.
INTERPRETATION
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 <>
3. Colony count of 5,000 to 50,000 CFU/ml (10 to 50 colonies)
a. If mixed: Final report: “Multiple microorganisms present: probable contamination; please repeat culture”. NOTE: A colony count is reflective of one organism. Each organism has its own colony count. Do not use a total cumulative count when more than one organism is isolated.
b. If pure culture of one or two potential pathogens equally predominant
· For patients on antimicrobial agents, female patients with urethritis, and symptomatic males, enumerate and 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.
· If no clinical information is provided, enumerate and provide a descriptive identification of colony morphology for organism present. Hold the culture at room temperature for possible further workup if requested by medical provider.
4. Colony count 50,000 to >100,000 CFU/ml (more than 50 colonies)
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.
PROCEDURE NOTES
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.
IDENTIFICATION
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>
2. If growth occurs on both plates and the colonies are morphologically similar, then it is safe to assume the organism is of the Enterobacteriaceae family (gram negative). Identification may be made using the API test procedure. Perform antibiotic sensitivity.
3. Quick ID’s: The colony morphology and spot indole test can be used to make presumptive identifications of E. coli, Klebsiella-Enterobacter spp., and Proteus (Refer to Spot Indol procedure in SOP). Refer to flowcharts found in “Processing Culture” procedure and/or Table 1.
· E. coli – Will have a flat pink colony morphology on MAC, and will sometimes be beta hemolytic on BA. E.coli will also be indole positive. If the isolate has these characteristics and a typical smell and colony morphology the isolate may be called an E.coli.
· Proteus spp. – Produce a foul odor and tend to produce a film of swarming growth that covers the surface of the BA plate. On MAC, these organisms are non-lactose fermenters colonies (colorless) and may be either indole-positive or indole-negative when tested from blood agar. If indole positive, it can be called P. vulgaris. If indole negative, it can be called P. mirabilis.
· Klebsiella spp. and Enterobacter spp. – Usually large, mucoid, and pink on MAC. These species are usually spot indole negative.
4. Quick ID - Pseudomonas aeruginosa – Will be a non-lactose fermenter on MAC and will have a very typical colony morphology on BA. On BA the organism may or may not b beta hemoloytic, it will have blue or green pigment, “grape-like” odor, and a spreading colony type. The isolate will also be oxidase positive. If the organism has these characteristics, it can be called a P. aeruginosa.
5. If there is growth on the BA but not on MAC, a gram stain should be done on the organism to determine its gram reaction. See Table 2.
6. For Enterococcus sp., susceptibilities should not be done for outpatients. Simply report the drugs of choice and hold the plates for 3 days. If heavy growth on a repeat culture, a sensitivity can probably be done on a MH plate. Drugs of choice are for an uncomplicated UTI is ampicillin. Alternates are nitrofurantoin and levofloxacin.
7. Gram-positive rods frequently isolated but rarely involved in urinary tract infections are Corynebacterium sp. (diphtheroids) and Lactobacillus sp. These organisms will appear as tiny, nondescript colonies on BA and will not grow on MAC.
8. When yeast are isolated that have “spiking” or “feet” around the colonies, this will give a presumptive identification of C. albicans. For yeast that are very tiny on the BA (even after two days incubation) and the individual yeast cells are quite small (by gram stain or wet prep), a presumptive identification can be determined for Torulopsis glabrata. If possible, differentiate between the two yeasts; it could be clinically significant and very helpful to the medical provider. Report only when the amount is moderate to many. Report the yeast only when the amount is moderate-many.
9. Report any amount of Group B streptococcus isolated on women known to be pregnant or on any woman of child bearing age.
LIMITATIONS
1. Because most antibiotics concentrate in urine, negative urine cultures are possible following treatment of a urinary tract infection by an antibiotic that is only partially effective. For this reason, urine cultures collected to determine whether a course of therapy was effective should be collected 7 days after discontinuance of the antibiotic.
2. Improper specimen collection and/or allowing the urine to remain at room temperature for extended periods of time results in false positive cultures. The laboratory can prevent issuance of false positive reports, to a degree, by rejecting polymicrobial specimens.
3. Failure to obtain separation of colonies on primary plating will produce erroneous colony counts.
REFERENCE
1. Barry, A.L. et al. Cumitech 2. Laboratory diagnosis of urinary tract infections. Washington, American Society for Microbiology, 1975.
2. Baron, E.J., and S.M. Finegold. Bailey and Scott’s Diagnostic Microbiology, 8th ed. St. Louis, C.V. Mosby Co., 1990.
3. Clarridge, J.E., Johnson, J.R., Pezzlo, M.T. 1998. Cumitech 2B, laboratory Diagnosis of Urinary Tract Infections, Coordinating ed., A.S. Weissfield. ASM, Washington, D.C.
4. Miller, J.M.: B.B. Wentworth. Methods for quality control in diagnostic microbiology. APHA, Washington, D.C., 1985, pp. 89-134.
5. Miller, J.M. Handbook of Specimen Collection and handling in Microbiology. Atlanta, Centers for Disease Control, PHS, HHS, 1985.
6. Murray, P., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolken, R.H. 1999. Manual of Clinical Microbiology, 7th Edition. The ASM Press, Washington, D.C.
