Urine Sample Collection and Processing for the Diagnosis of UTI

Urine samples should be collected and sent for culture only when there is strong evidence/suspicion of urinary tract infections to avoid detection of asymptomatic bacteriuria.

Common symptoms of UTI are:

Frequency of urination

Burning sensation during urination


Dysuria (painful urination)

Pain above the pubic region

Cloudy, bloody or strong-smelling urine

Flank pain or pain above pubic area

Urine collection bottle (urinary tract infection)

Both noninvasive and invasive methods for collecting urine samples are available. Samples obtained from invasive methods are reliable as they are less likely to be contaminated and easy to interpret.

Urine collected by noninvasive methods passes through the contaminated milieu, so quantitative culture is used to diagnose urinary tract infections (UTI) and to discriminate between contamination, colonization (asymptomatic bacteriuria), and infection.

Positive cultures could mean
Colonization or

Commonly used methods for urine specimen collection

Suprapubic aspiration

Suprapubic aspiration is the best method to avoid contamination of specimens with bacteria in the distal urethra. Urine is withdrawn directly into a syringe through a percutaneously inserted needle.  This type of collection technique may be indicated in pediatric practice.

Use: Used infrequently because it is not indicated clinically (except in rare circumstances). It is invasive and uncomfortable and requires too much time and resources to be practical.

Straight catheter technique

Collection of urine by use of a single catheter (straight catheter technique) is the next-best technique for obtaining urine specimens with minimal contamination. It is an invasive technique with added disadvantages because inserting a catheter through the urethra can introduce bacteria into the bladder (and thereby cause UTI), and rare complications have been reported.

Use: It is not indicated clinically for most patients because it is too labor-intensive and costly for routine use.

Clean Catch Midstream Urine

It is the least invasive technique and is used widely. It has an obvious disadvantage compared to the techniques mentioned above. The chances of contamination from normal vaginal, perineal, and anterior urethral flora are high. The urine sample passes through the distal urethra and can become contaminated with commensal bacteria.

Commensal flora (resident flora) found in the urine samples are;

  1. Anaerobic cocci
  2. Anaerobic gram-negative bacilli
  3. Coagulase-negative staphylococci (excluding S. saprophyticus)
  4. Commensal Mycobacterium spp.
  5. Commensal Mycoplasma spp.
  6. Diphtheroids (Corynebacterium spp.)
  7. Lactobacilli
  8. Nonpathogenic Neisseria spp.
  9. Propionibacterium spp.
  10. Viridans and non-hemolytic streptococci

Use: Most urine specimens are obtained from adult patients via the clean-catch midstream technique

How to collect Midstream Specimen of Urine (MSU)?

Steps to collect a midstream urine specimen for Females

  1. Remove undergarments.

  2. Wash hands thoroughly with soap and water, rinse them, and dry them on a disposable paper towel or shake off excess water.

  3. Spread labia, with one hand, and keep them continuously apart.

  4. Open the WASH PACK and wash the urinary opening and the surrounding area. Discard the cloth in the wastebasket.

  5. Take the open sterile cup in the other hand without touching the rim or inner surface of the cup or lid.

  6. Void 20 to 25 ml into the toilet and catch a portion of the rest of the urine in the container without stopping the stream.

    Do not touch the legs, vulva, or clothing with the cup.

  7. Place the lid securely on the cup.

  8. Immediately transfer to the microbiology laboratory or follow the procedure as indicated by the hospital personnel.

Instructions for male patients

  1. Remove undergarments.
  2. Wash hands.
  3. Retract the foreskin completely.
  4. Wipe the head of the penis in a single motion with the first towelette. Repeat with a second towelette. If not circumcised, hold foreskin back before cleansing.
  5. Void 20 to 25 ml into the toilet and catch a portion of the remaining urine in the cup without stopping the stream. Do not touch the cup with the penis.
  6. Place the lid on the cup securely
  7. Immediately transfer to the microbiology laboratory or follow the procedure as indicated by the Hospital personnel.

Indwelling Catheter

Specimen collection from patients with indwelling catheters requires a scrupulous aseptic technique. The catheter tubing should be clamped off above the port to allow the collection of freshly voided urine. The catheter port or wall of the tubing should then be cleaned vigorously with 70% ethanol, and urine aspirated via a needle or syringe; the integrity of the closed drainage system must be maintained to prevent the introduction of organisms into the bladder.

Related Post: Microbial Etiology of Urinary Tract Infections (UTI)

Processing of Urine Sample


The standard urine culture protocol uses 1 μl (0.001 mL) of urine, spread quantitatively onto 5% sheep blood agar plate and MacConkey agar, and incubated aerobically at 35°C for 24 h.

  1. A loopful of well-mixed midstream urine samples is inoculated into the blood agar plate, MacConkey agar plate and CLED (Cysteine Lysine Electrolyte Deficient agar) or using the semi-quantitative method (i.e with the help of the calibrated loop of 4 mm size)
  2. A loopful of the urine is touched to the center of the plate from which the inoculum is spread in diameter across the plate. Without flaming or reentering the urine, a loop is drawn across the entire plate, crossing the first inoculum streak numerous times.
  3. The Blood Agar plate and CLED or Mac-Conkey agar plate are both incubated aerobically at 37°C for 24 hours.
  4. The following day the plates are observed for the growth of the organism, their characteristics on the plates, or any possible contamination.

Interpretation of the result:

  1. Less than 104 organisms/ml: not significant
  2. 104– 10organisms/ml: doubtful significance (suggest repeat collection)
  3. More than 105 organisms/ml: significant bacteriuria

Only samples showing significant growth are further processed.


In certain conditions, even the growth between 103-10is also considered significant. Those conditions are:

  • Patient is on diuretics
  • Patients being on antimicrobial

In certain conditions, even any count is considered significant. Those conditions are

  • Specimen obtained from catheter tubing
  • Suprapubic aspirate
  • Suspected hematogenously acquired  infection (eg. Staphylococcus  aureus)


  1. Llor, C., Moragas, A., Aguilar-Sánchez, M., García-Sangenís, A., Monfà, R., & Morros, R. (2023). Best methods for urine sample collection for diagnostic accuracy in women with urinary tract infection symptoms: a systematic review. Family practice, 40(1), 176–182. https://doi.org/10.1093/fampra/cmac058 
  2. Karacan, C., Erkek, N., Senel, S., Akin Gunduz, S., Catli, G., & Tavil, B. (2010). Evaluation of urine collection methods for the diagnosis of urinary tract infection in children. Medical principles and practice : international journal of the Kuwait University, Health Science Centre, 19(3), 188–191. https://doi.org/10.1159/000273068
  3. Sinawe H, Casadesus D. Urine Culture. [Updated 2023 May 1]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557569/

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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