Urine is a sterile fluid and can become easily contaminated with micro biota from the perineum, prostrate, urethra, or vagina. It is important that urine is collected according to the instructions provided to ensure proper specimen collection.
Epidemiologic studies have shown that bacterial count of >or equals to 105 CFU/ml for a pure culture of gram negative bacilli were found to associated for acute bacterial infection of urinary tract. In females with dysuria and acute UTI , others reported as >102 CFU/ml can be significant whereas in infants and catheterized patients ,low count also have been shown to be significant.
The etiological agents of urinary tract are generally limited to patients own intestinal micro biota with E.coli ,Enterococcus species, Klebsiella species, Enterobacter species, and Proteus species.
- Repeat urine specimen where there is no evidence of refrigeration and the specimen is >2 hr old.
- Reject 24 hr urine collection.
- Reject Foley catheter tips as unacceptaqble for culture, they are unsuitable for the diagnosis of urinary infection.
- Reject the urine from the bag of the catheterized patient.
- Except for suprapubis specimen , reject specimen for anaerobic culture.
For voided urine specimen and those of indwelling catheter culture at least 0.001ml.
For other urine specimen , culture 0.01ml.
INNOCULATION METHOD FOR COLONY COUNT
Calibrated loop method
Using the disposable or flamed and cooled calibrated loo , hold the loop vertically and immerse it just below the surface of a well-mixed uncentrifuged urine specimen.
Deliver a loopful of well mixed urine onto the blood agar plate or CLED and spread using one o the three methods below;
- Using the loop, make a straight line down the center of the plate and streak the urine by making the series of passes at 90 degree angle through the inooculum (method for 0.001ml only ) .
- Deliver the loopful onto one quadrate of plate by making a straight line or a V shaped line. Streak urine onto the first quadrant, and proceed by streaking onto all four quadrants.
- Spread the loop ful urine over the surface of the entire plate, using the loop in three directions or a sterile spreader or bend rod (hockey stick)
Incubate in ambient air overnight 35 to 37OC
- Perform anaerobic culture only on anareobically collected suprapubic bladder aspirate (received in a syringe) on special request or when bacteria suggestive of anaerobes are seen in the direct of smear but fail to grow culture.
- If convenient, incubate BAP and CAN in 5 % Co2 to enhance growth of gram positive organism.
Examination of culture media
- Examine culture that have been incubated overnight but make final reading at 18 h unless the specimen fits the criteria below.
- Reincubate until culture has incubated for 48 h if one of the following is true
- The specimen was collected by an in invasive technique, such as suprapubic bladder aspiration or straight catheter method.
- Tiny or scant colonies are present that are barely discernible
- Culture result do not correlate with gram stain findings or clinical conditions(eg, the patient has sterilize pyuria or symptoms of positive culture)
- The patient is immune compromise including patients who has transplanted organs
- Yeast or fungal culture is requested or appropriate ( eg, neonatal intensive care unit culture)NOTE Incubation may need to be upto 72 h if CNA or EMB is not used. Many yeast grow will on EMB.
c) For positive culture , examine culture media for the quantity and morphological type of organism present .
1) With a 0.001 ml loop , one colony equals 1000 cfu/ml
2) With a 0.01ml loop , one colony equals 100 cfu/ml
3)When the colony are too numerous to count the maximum readable using the 0.001 ml loop
is > 10 5 cfu/ml and the maximum readible on the 0.01 ml loop > 104 cfu/ml
Clinical microbiological procedures handbook. Henry.D.Isenberg