Urine culture بسم الله الرحمن الرحيم Diagnostic Medical Microbiology-Laboratory Manual

Routine Urine Culture Aim of the test : An etiological diagnosis of bacterial urinary tract infection by semi quantitative cultivation of the urine with identification and susceptibility test of the isolated bacteria(s). Types of specimen: Urine (Midstream urine), suprapubic aspiration, catheterized urine. Note: First morning specimens yield highest bacterial counts from overnight incubation in the bladder, and are the best specimens. Criteria of specimen rejection : Un-refrigerated specimen older than 2 hours may be subject to overgrowth and may not yield valid results; unlabeled specimen; mislabeled specimen; specimen in expired transport container; 24 hours urine specimens.

*Staphylococcus aureus Pathogens and commensals : Common pathogens Commensal flora Neisseria gonorrhoeae any colony on chocolate or TM agar (special request). Diphtheroid bacilli E.coli and other Enterobacteriaceae Lactobacillus spp Enterococcus spp Coagulase negative Staphylococci *Staphylococcus aureus Pure culture regardless to the no. of CFUs. Alpha Haemolytic Streptococci Staph saprophyticus Bacillus spp Corynebacterium jeikeium Non pathogenic Neisseria spp. Acinetobacter spp Anaerobic cocci Pseudomonas spp Commensal Mycobacterium * Gardnerella vaginalis Unusual Commensal Mycoplasma spp. Beta -haemolytic streptococci * Salmonella spp (early stage of infection) * yeast Parasites Schistosoma haematobium Trichomonas vaginalis

Pre specimen processing Patient preparing: Instruct the procedures for the patient Specimen collection Collection of midstream urine for bacterial investigation: Patient not needing assistance: Give the patient a suitable container. Instruct the patient before the collection, preferably with illustration. Tell the patient not to touch the inside or rim of the container.

Pre specimen processing Who will collect the specimen: Midstream urine is collected by the patient. If disabled, nursing staff will assist in collection. For catheterized specimen, nursing staff will collect the specimen. Suprapubic aspiration is performed by the physician. Quantity of specimen : To fill line in transport tube (~20 mL). Time relapse before processing the sample : The maximum time allowed for processing a urine sample is 2 hours from the time of collection. Storage : At room temperature unless delay is inevitable; it must be refrigerate or mixed with preservative like boric acid. The conditions and concentrations of urine preservatives are defined for 24-hour collections as follows:   Ambient Room temperature (21°-25° C) Refrigerated 4° C Frozen -20° C 6N HCl 30 mL per 24-hour collection Acetic Acid 50% 25 mL per 24-hour collection Na2 CO3 (crystals) 5 g per 24-hour collection Toluene 30 mL per 24-hour collectioN 6N HNO3 15 mL per 24-hour collection Boric Acid 10 g per 24-hour collection Thymol (10% in isopropanol) 10 mL per 24-hour collection Suprapubic aspiration

Specimen processing

Initial report The use of dipstick designed to detect the presence of urine nitrite and to indirectly estimate the number of segmented neutrophiles through the detection of leukocyte esterase activity. Rationale for the nitrate test is that most urinary tract infections are caused by nitrate reducing members of the family Enterobacteriaceae. Leukocyte esterase (LE) is produced by segmented nutrophiles; this test when performed alone correlated with ten or more white blood cells per high power field in the urine with a sensitivity in the range 88% and specificity 94%. Pyuria refers to urine which contains pus. Defined as the presence of 4 or more neutrophils per high power field.

Pyuria: the increased number of WBC in urine sample. sterile pyuria: is a condition arises when there is an elevated in WBC in urine and negative culture.

Screening test A drop of well-mixed urine is allowed to air dry. As many as 60% to 80% of all urine specimens received for culture by the acute care medical Center laboratory may contain no etiological agents of infection. Procedure developed to identify quickly those urine specimens that will be negative on culture, thus to circumvent excessive use of media, technologist time, and the overnight incubation period. The gram stain is the easiest, least expensive, and probably the most sensitive and reliable screening method for identifying urine specimens that contain greater than 10^5 CFU/ml. A drop of well-mixed urine is allowed to air dry. The smear is stained and examined under oil immersion (1000x). Presence of at least one organism per oil immersion field. ( examining 20 fields ) corelates with significant bacteriuria (>10^5 CFU/ml).

Specimen processing Culturing:

Culturing Procedure: Mix the urine sample to re-suspend microorganism present. Dip a 1 μl or 10 μl calibrated loop in vertical position in the urine and remove the loop and use the collected fluid to inoculate Nutrient, Blood and MacConkey agars respectively.

Culturing Procedure:

Colony counting A plate count of 100,000 CFU/ml of pure culture should be considered positive and isolated organism should be identified and sensitivity test will be performed. A plate count between 10,000 – 100,000 CFU/ml is considered suspected . A plate count less than 10,000 CFU/ml is considered negative.

Colony counting Catheterized urine: Plate count > =1000 CFU/ml Suprapubic urine : Any colony is significant Routine urine culture(voided urine specimen): Plate count >=105 CFU/ml

Post specimen processing Interfering factors: Patient on antibiotic therapy. Improper sample collection. Result reporting: Report wet mount as an initial report. Report the isolated pathogen and its sensitivity pattern as a final report. Turn around time: Wet mount results should be available 1 hour after specimen receipt. Isolation of a possible pathogen can be expected after 2-3 days. Negative culture will be reported out 1-2 days after the receipt of the specimen.

Additional information A single culture is about 80% accurate in the female; two containing the same organism with count of 10^5 or more represents 95% chance of true bacteriuria; three such specimens mean virtual certainty of true bacteriuria. Single clean voided specimen from an adult male may be considered diagnostic with proper preparation and care in specimen collection. Urinary tract infection is significantly higher in women who use diaphragm-spermicide contraception, perhaps secondary to increased vaginal pH and a higher frequency of vaginal colonization with E. coli. If the patient is receiving antimicrobial therapy at the time the specimen is collected, any level of bacteriuria may be significant. Failure to recover aerobic organisms from patients with pyuria or positive Gram's stains of urinary sediment may indicate the presence of mycobacteria or anaerobes.

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