Effective Date: 
Wed, 08/07/2013
Tue, 01/10/2017
Wed, 08/07/2013

Purpose and Scope:

Urinary tract infections (UTI) are one of the most commonly encountered acute infectious diseases. Most UTIs occur as a result of bacteria ascending the urethra and entering the urinary bladder.
Urine specimens for culture are collected when the following syndromes are suspected:
cystitis, pyelonephritis, asymptomatic bacteriuria, and less commonly acute prostatitis, pyelonephric abscess, and urosepsis.
Among the bacteria most commonly isolated from patients with acute uncomplicated cystitis are Escherichia coli, Klebsiella species, and other Enterobacteriaceae and Staphylococcus saprophyticus.
Group B Streptococcus are markers of colonization in pregnant women.

Principle of the method:

Urine cultures are evaluated with consideration given to factors such as colony count, type and number of organisms growing, type of specimen and the clinical condition of the patient.


Setup Procedure:

1. Bring BAP/EMB plates to room temperature.
2. Ensure urine is well mixed and uncentrifuged.
3. Hold plastic 1 uL calibrated loop vertically and immerse it just below the surface of the urine.  Ensure that urine completely fills the loop.
4. Spread the loopful of urine first on the BAP. Streak plate using the Quantitative method as in the diagram below.
5. Redip the loop in the urine and spread on the EMB. Streak plate using the Semi-Quantitative method as in the diagram below.

 6. Incubate aerobically at 35°C

Negative Culture Examination

Examine cultures that have been incubated overnight.
If there is no visible growth at 24 hours, send a final report“No growth ”.

Positive Culture Examination

Cultures with growth:

Count the colonies and multiply by the appropriate dilution factor in SI units.
• One colony equals 1,000 col/ml with the 0.001 ml loop (blue). (1000 x 103CFU/L)
• One colony equals 100 col/ml with the 0.01 ml loop (yellow). (100 x 103 CFU/L)
Colony count is evaluated by individual isolate, not combined growth.

 Single organism:

 1000-10,000 col/ml of a pathogen Ask MD if workup needed
 >10,000 col/ml of a potential pathogen       ID and sensitivities to be performed
 >100,000 non-pathogen     (Lactobacillus,diptheroids,Strep viridans, Coag-neg Staph) No sensitivities. Report ID only.


Two organisms:

Both potential pathogens and >100,000 col/ml                ID and sens X2
 One potential pathogen >100,000 and predominate.      ID and sens X1, report minority 
        bug as urinary flora
No predominate potential pathogen (any count)   Report mixed urinary flora


More than two organisms:

One potential pathogen >100,000 and predominate*. ID and sens X1, report minority bugs as mixed urinary flora
*(predominate is > 2x other organisms)  

Organism identifications

1. If organism is suggestive of E. coli morphology (i.e.-lactose fermenter,greenish   metallic sheen on EMB) perform an indole test and, if positive, report as: “ > xxx col/ml presumptive E. coli)
2. Other oxidase negative gram negative rods may be identified using the Microgen GN ID system. See individual Microgen GN ID procedure for further instructions.
3. Oxidase positive gram negative rods should be sent to Quest on a blood agar slant for ID and sensitivities.
4. Do not routinely perform definitive identification on any yeast with growth at any quantity.  Report out, i.e. “>xxx cfu/mL Yeast.  Notify Microbiology if further ID is needed.”
5. Organisms that grow only on BAP side of plate should be tested by wet mount or gram stain to ensure correct workup algorithim is invoked.
6. Gram positive cocci that are catalase positive should be tested by tube coagulase method.

7. Coagulase negative staphylococcus should be tested for Novobiocin resistance procedure.  Any  isolate determined to be Staphylococcus saprophyticus should have the comment, “Infections respond to concentrations achieved in urine of antimicrobial agents commonly used to treat acute, uncomplicated urinary tract infections (eg.nitrofurantoin, trimethoprim+sulfamethoxazole or a fluoroquinolone”.

8. Coagulase positive (Staph aureus) should be sent to Quest on a blood agar slant for sensitivities.

9. Gram positive cocci that are coagulase negative (Streptococci) should be observed for beta hemolysis.

10. Beta hemolytic organisms can be tested with CAMP method for Group B identification (Strep agalactiae) and Rapid Strep for Group A (Strep pyogenes).

11.  Streptococci that are CAMP neg and/or Rapid Strep neg , beta hemolytic colonies can be reported as Beta Hemolytic Strept not Group A or B.

12. Non-hemolytic catalase negative GPC should be tested with PYR for Enterococcus sp rule out. If PYR positive the Enterococcus should be sent to Quest on a blood agar slant for sensitivities.

13. Any organisms which are thought to be clinically significant and cannot be identified with the available methods should sent to Quest on a blood agar slant for further workup.


Key Points: 

Specimen Requirement:

Clean catch, midstream urine collected in plastic, non-sterile or sterile container.
Specimen should be plated within 1 hour of collection or may be refrigerated up to 24 hours.
Specimen does not need to be brought to room temp before plating.

Quality Control:

Perform Quality Control as required for each individual identification method used in evaluating and testing. See procedures for identification and susceptibility testing.

Reagents and Supplies:

Sheep's blood agar plates

EMB agar plates
Calibrated inoculating loop (disposable)
37 C Incubator


Forbes, Betty; Sahm, Daniel; Weissfeld, Alice; Bailey and Scott's Diagnostic Microbiology, 10th Edition, 1998.

Isenberg, Henry; Essential Procedures for Clinical Microbiology, Edition 1998.