Urine Culture Techniques

Normally, urine is sterile when excreted by the kidneys and stored in the urinary bladder. When it is voided, however, urine becomes contaminated by the normal flora of the urethra and other superficial urogenital membranes. The presence of bacteria in voided urine (bacteriuria), therefore, does not always indicate urinary tract infection. To confirm infection, either the numbers of organisms present or the species isolated must be shown to be significant.

Active infection of the urinary tract arises in one of three ways:

1. microorganisms circulating in the bloodstream from another site of infection are deposited and multiply in the kidneys to produce pyelonephritis by the hematogenous (originating from the blood) route;
2. bacteria colonizing the external urogenital surfaces ascend the urethra to the bladder, causing cystitis (infection of the bladder only) or pyelonephritis by the ascending route; or
3. microorganisms, usually from the urethra, find their way into the bladder on catheters or cystoscopes.

Cystitis is much more common than pyelonephritis. In the former case, most of the offending organisms are opportunistic members of the fecal flora for example, E. coli (by far the most frequent cause of urinary tract infection), Klebsiella, Enterobacter, Serratia, and Proteus. Pseudomonas and enterococci are also often incriminated, especially in hospitalized patients with indwelling urinary catheters or those receiving multiple antimicrobial agents. When these nonfastidious organisms reach the bladder, where active host defense mechanisms (blood phagocytes and antibodies) are not readily available, they may grow in the urine, producing acute bladder and urethral symptoms (urgency; frequent, painful urination).

The blood that flows through the kidneys normally carries no microorganisms because phagocytic white blood cells and serum antibodies are constantly at work eliminating any microbial intruders that reach deep tissues. If these defense mechanisms are not working well or become overwhelmed by extensive infectious processes in systemic tissues (uncontrolled tuberculosis or yeast infections, staphylococcal or streptococcal abscesses), then the kidneys may become infected by organisms carried to them via the bloodstream. More commonly, however, microorganisms initially colonizing the bladder ascend the ureters to infect the kidneys.

Laboratory diagnosis of urinary tract infections is made by culturing urine, usually obtained either by catheterization or by voided collection. To obtain a catheterized urine specimen, a sterile, polyurethane catheter tube is inserted into the urethra and passed up into the bladder. The urine drains through the catheter tube and is collected in a sterile specimen cup. If it is obtained properly, this sample represents urine obtained directly from the bladder. Catheterized urine is not contaminated by normal urogenital flora, but the technique itself may introduce organisms into the bladder. For this reason, catheters are seldom used to collect routinely ordered urine cultures. In culturing voided specimens, however, the laboratory is faced with several problems. One is the normal contamination of voided urine; another is the need for speed in initiating culture before contaminants can multiply and distort results; and a third is the obligation to obtain and report results that reflect the clinical significances of the isolates adequately and accurately. Contamination by hardy, nonfastidious organisms can mask the presence of other pathogens that are difficult to cultivate on artificial media. Overgrowths in standing urine give a false picture of numbers. Either situation can lead to laboratory results that fail to reveal the clinical problem, and possibly to the mismanagement of the patient’s case.
To address these problems, the laboratory must insist on proper techniques for urine collection and on prompt delivery of specimens for culture. When delay is unavoidable, urine specimens should be refrigerated to prevent multiplication of any microorganisms they may contain. Alternatively, a novel urine transport system that inhibits the growth of bacteria in urine without refrigeration has been developed. The system consists of a sterile evacuated tube that contains boric acid. Once the urine sample is collected, it is aspirated immediately into the evacuated tube. The boric acid, which is nontoxic to bacteria, disperses throughout the urine and inhibits bacterial growth in the sample for up to 12 hours at room temperature. Upon receipt in the laboratory, the urine is examined for certain physical properties that can indicate infection, for example, color, odor, turbidity, pH, mucus, blood, or pus. Uncontaminated urine is usually clear, but sometimes may be clouded with precipitating salts. Urine containing actively multiplying bacteria is turbid. If the patient has a urinary tract infection, the urine usually also contains many white blood cells. In some instances, the mere recovery of a pathogenic bacterial species in urine (e.g., Salmonella, Mycobacterium tuberculosis, or beta-hemolytic streptococci) is significant, regardless of numbers, and the search for such organisms does not require quantitative culture technique. It is generally advisable, however, to culture urine quantitatively, and to report a “colony count”—that is, the numbers of colonies that grow in culture from a measured quantity of urine. If microorganisms are actively infecting the kidneys or bladder, they can usually be demonstrated in large numbers in urine (in excess of 100,000 organisms per milliliter of urine). The recovery of greater than 100,000 bacteria per milliliter of urine in a properly collected and transported urine specimen is referred to as significant bacteriuria because the presence of such large numbers of bacteria in urine correlates with active infection of the bladder or kidney. On the other hand, normal urine that is merely contaminated in passage down the urethra contains very few organisms (100 to 1,000 per milliliter, not more than 10,000), provided it is cultured soon after collection, before multiplication of contaminants can occur in the voided specimen awaiting culture. Some patients with symptoms of cystitis have low counts of the causative agent in their urine and close collaboration between the laboratory and the physician is needed to accurately diagnose these infections.

Collection of voided urine for culture (“clean-catch” techniques)
Aseptic urine collection requires careful cleansing of the external urogenital surfaces, using gauze sponges moistened with tap water and liquid soap.

For males, the procedure simply entails thorough sponging of the penis, discard of the first stream of urine, and collection of a “midstream” portion in a sterile container fitted with a leak-proof closure. If the outside of the container has been soiled with urine in the process, it must be wiped clean with disinfectant before being handled further.

For females, extra care is necessary. All labial surfaces must be thoroughly cleansed, and the sterile container must be held in such a way that it does not come in contact with the skin or clothing. Again, the first stream of urine is discarded, and a midstream sample is collected. When the container has been tightly closed, it is wiped clean with disinfectant.

Urine containers should never be filled to the brim. Closures should be double-checked to make certain they will not permit leakage during transport to the laboratory. If there is any delay (before or after delivery to the lab) in initiating culture, urine specimens must be refrigerated.