Answering your questions

Dec. 1, 2010

Urine collection, preservation, and transport

We collect our urine cultures in Boritex vials, but some accounts insist on collecting in sterile cups. What is the stability of urine in a sterile cup for culture? Have there been any recent data that supports which is best for urine preservation and transport?

AUrine culture with susceptibility testing is an essential part of the laboratory diagnosis of urinary-tract infections (UTI). Gram-negative bacilli, especially Escherichia coli, are the main bacteria isolated from these infections. In a pure culture, counts of ≥ 105 colony forming units (CFU) of Gram-negative bacteria per milliliter of urine are necessary for diagnosis. In mixed cultures, however, clinical microbiology laboratories follow certain rules for selective identification and reporting. Bacteria grow well in urine; and, sometimes, it can be contaminated with urethral, skin, genital, or fecal flora. Contaminating bacteria are usually present in low numbers (<104 CFU/mL), but can reach significant levels with prolonged incubation (> 2 to 4 hours) at room temperature, rapid growth rate of the organism, and increased initial degree of contamination. The Clinical and Laboratory Standards Institute (CLSI) recommends urine culture within two hours of collection1. Midstream urine specimens are recommended for microbiological culture and antimicrobial-susceptibility testing because of reduced cellular and microbial contamination compared to initial urine specimens. Patients should adhere to the instructions provided by healthcare professionals for urine collection.

When the transport of urine samples to the microbiology laboratory is expected to be delayed for more than two hours, samples should be refrigerated, because urine is stable for 24 hours in this environment. Unfortunately, sometimes the refrigeration of urine samples is difficult, especially if they are collected off site. Under such circumstances, preservation of urine is necessary to provide a stable environment for the specimen and prevent bacterial overgrowth (similar to refrigeration) until testing can be conducted. The most commonly used preservative is boric acid. This preservative may be used in tablet, powder, or lyophilized form. Preserved urine specimens can be stored at room temperature for 48 to 72 hours before processing. When preservatives are used, the correct specimen-to-preservative ratio must be maintained. Use of the indicated fill lines on the tubes can assist with ensuring the correct fill volume. Underfilling the tube will lead to a high concentration of preservative in the specimen leading to inhibition of subsequent bacterial growth, while over-filling the tube will overly dilute the preservative resulting in underestimation of bacterial counts. Nickander, et al, studied the effect of urine volume on the results of culture in two commercial systems that use boric acid as a preservative.2 It was concluded that if the volume of urine used is less than the recommended volume, the growth of E coli and Klebsiella pneumoniae was inhibited if examined at four or 24 hours. When enough urine was used, however, the results were the same as that of the starting urine at four hours but different in 24 hours.

The results of studies examining the effect of preservatives on urine culture results are conflicting. These differences are believed to be due to delayed incubation, different preservative preparations, and different urine volumes. Weinstein compared the culture results between a transport kit containing a lyophilized preservative with those of a conventional sterile cup without preservative. Overall agreement of results of 853 cultures after initial processing of samples in the laboratory was 98.7%. Results of 320 repeat cultures after a 24-hour holding period (preserved samples were left at room temperature while unpreserved samples were refrigerated) and 277 repeat cultures after a 48-hour holding period also showed excellent agreement (99.1% and 95.7%, respectively.3 Similar results were observed by Lum, et al,4 In another study, however, comparing the results of culture at initial processing and after overnight culture of the same preserved specimens, significant alterations were observed in 16%, and the clinical interpretation differed in 8% of the specimens.5. This study did not address the effect of refrigeration on overnight incubation of sterile cups.

For urine samples that can be transported to the laboratory within two hours, both sterile urine cups and tubes with preservatives can be used. If the volume of collected urine is below the recommended amount by the manufacturer (about 3 mL to 5 mL), sterile cups are better due to the concentration of the preservative and its inhibitory effect on bacteria. When delay in transporting specimens is expected (>2 hours), refrigeration or the use of tubes with preservatives is recommended. To decrease the potential effect of preservatives, enough volume of urine should be collected and specimens should be processed in 24 hours. Unpreserved specimens exceeding the two-hour limit that have not been refrigerated should not be accepted for analysis due to potential bacterial overgrowth and invalidation of bacterial colony counts. Different time limits of collection and effects of preservatives exist for molecular tests of urine specimens.


  1. Clinical and Laboratory Standards Institute. Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved Guideline – Second Edition. Wayne, PA. Clinical and Laboratory Standards Institute; 2001.CLSI document GP-16A2.
  2. Nickander KK, Shanholtzer CJ, Peterson LR. Urine Culture Transport Tubes: Effect of Sample Volume on Bacterial Toxicity of the Preservative. JCM. 1982;15:593-595.
  3. Weinstein MP. Clinical Evaluation of a Urine Transport Kit with Lyophilized Preservative for Culture, Urinalysis, and Sediment Microscopy. Diagn Micr Infec Dis. 1985;3:501-508.
  4. Lum KT, Meers PD. Boric Acid Converts Urine into an Effective Bacteriostatic Transport Medium.
    J Infec. 1988;18:51-58.
  5. Gillespie T, Fewster J, Masterton RG. The Effect of Specimen Processing Delay on Borate Urine Preservation. J Clin Pathol. 1999;52:95-98.

—Adnan Alatoom, MD, PhD
Department of Laboratory Medicine and Pathology
Mayo Clinic Rochester, MN

Urine colony counts for pediatric patients

Q What is the procedure for colony counts on pediatric patients. Assuming the urine is collected properly (e.g. catheter), are all colonies worked up? Is there a quantitation formula?

A The definition of significant bacteruria is >105 colony-forming units (CFU)/mL of urine from a clean-catch specimen. If the urine is collected by using a urethral catheterization, a significant colony count is defined as >50,000 CFU/mL (or less strictly, >10,000 CFU/mL).1 Those specimens collected by suprapubic aspiration should not be contaminated and any amount of bacteria is considered significant, although most samples will contain at least 105 CFU/mL.1 For all urine collections, lower quantities of bacteria usually are the consequence of contamination of the specimen.

—Susan E. Sharp, PhD, D(ABMM)
Director of Microbiology,
Kaiser Permanente Pathology Regional Laboratory;
Associate Professor Oregon Health and
Science University Portland, OR


  1. Ward ER. Cystitis and pyleonephritis. In Textbook of Pediatric Infectious Diseases. 5th ed. Philadelphia, Saunders, 2004;7:454-546.

Brad S. Karon, MD, PhD, is associate professor of laboratory medicine and pathology, and director of the Hospital Clinical Laboratories, point-of-care testing, and phlebotomy services at Mayo Clinic in Rochester, MN.

MLO's “Tips from the Clinical Experts” provides practical, up-to-date solutions to readers' technical and clinical issues from a panel of experts in various fields. Readers may send questions to Brad S. Karon, MD, PhD, by e-mail at [email protected].