Urine culture
D.M.M. Lab.
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.
Pathogens and commensals
Common pathogens
Commensal flora
Neisseria gonorrhoeae
any colony on
Diphtheroid bacilli
chocolate or TM agar (special request).
E.coli and other Enterobacteriaceae
Lactobacillus spp
Enterococcus spp
Coagulase negative Staphylococci
*Staphylococcus aureus
Alpha Haemolytic Streptococci
Pure culture regardless to the no. of CFUs.
Staph saprophyticus
Corynebacterium jeikeium
Acinetobacter spp
Pseudomonas spp
Bacillus spp
Non pathogenic Neisseria spp.
Anaerobic cocci
Commensal Mycobacterium
* Gardnerella vaginalis Unusual
Commensal Mycoplasma spp.
Beta -haemolytic streptococci
* Salmonella spp (early stage of infection)
Parasites
Schistosoma haematobium
Trichomonas vaginalis
* yeast
* Diagnostic Microbiology, BAILEY & SCOTT, 9th EDITION
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.
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: 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
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
CFU
# colonies counted
in original sample 
ml
(dilution) (volume plated, in ml)
 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.
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 diaphragmspermicide contraception, perhaps secondary to increased vaginal pH and a
higher frequency of vaginal colonization with E. coli.
 Most laboratories limit the number of organisms which will be identified
when recovered from urine to two.
 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.