Dr. med. Dirk Manski

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Urine Analysis (2/3): Urine Culture

Preanalytic Phase

Indications for urine culture in asymptomatic patients

Indications for urine culture in symptomatic patients:

Collection of specimen

First choice for the urine collection is during sterile catheterizations and interventions. Otherwise, the urine collection is made in men by midstream specimen, in women by catheterization or midstream clean-catch specimen (which is frequently contaminated). A midstream specimen in women has only a clinical significance, if it is not pathological. The urine collection in infants is made with an adhesive bag.

Four-glass test:

In order to identify a chronic bacterial prostatitis, four separate specimens (4-glass test) are collected and examined microbiologically with urine sediment microscopy and culture. Bacterial prostatitis is diagnosed if there is a 10-fold increase in bacteria between VB1/2 and EPS/VB3:

Due to the high costs and effort, a two-glass test has become standard, recent studies have confirmed the equivalence:

Transport of the specimen:

The examination of fresh urine within two hours is best. For longer transport the specimen must be cooled or a urine culture transport tube (containing boric acid) should be used.

Quantitative Urine Cultur

The colony count is determined with a quantitative urine culture. The most common used technique uses standardized plastic loops to distribute 10 μl urine on a agar plate (e.g. CLED-Agar) [fig. technique of quantitative urine culture]. The plates are incubated for 24 h at 36 degrees celsius. One colony equals 102 colony forming units per ml (cfu/ml), 10 colonies = 103 cfu/ml, 100 Kolonien = 104 cfu/ml and 1000 colonies = 105 cfu/ml. The colony count is determined either with counting or with a semiquantitative technique by comparing the agar plate with standardized figures.

figure technique of quantitative urine culture   

Semiquantitative urine culture: the primary inoculation of the plate is done with 10 μl urine using a standardized plastic loop. The specimen is distributed over the plate with a cross-streaking technique.


CLED is an unselective agar and often used for quantitative urine culture. The agar enables the growth of all typical bacteria of urinary tract infection. CLED stands for Cystine Lactose Electrolyte Deficient-Agar, the low concentration of electrolytes prevents swarming from proteus species. Bacteria with fermentation of lactose will produce yellow colonies and stain the agar yellow. Bacteria without fermentation of lactose (non-fermenters) will build grey to blue colonies [fig. bacterial colonies on CLED agar].

figure bacterial colonies on CLED agar   

Mixed culture of fermenting (yellow colonies) and non-fermenting (blue colonies) bacteria on CLED agar. Public domain figure from Microrao, JJMMC, Davangere, Karnataka, India, http://commons.wikimedia.org.

Significant bacteriuria:

≥ 105 colony-forming units in a midstream urine specimen speak in favour of a significant bacteriuria (Kass et al, 2002). In case of high diuresis, frequency or sterile collection of the urine specimen, even a lower bacteria count may represent a significant sign for urinary tract infection.

Identification of Bacteria

An identification of the bacteria makes sense if there is no mixed bacterial growth on the CLED agar plate (not more than two germs). Mixed bacterial growth suggests contamination during urine collection, the urine culture should be repeated with a new specimen. The correct identification of the bacteria will only be successful if isolated colonies are present on the agar plates. Mixed growth may remain unnoticed in confluent colonies. If in doubt, repeat streaking on a new agar plate to achieve isolated colonies [fig. streaking technique for the isolation of bacterial colonies].

figure Three-phase streaking technique to achieve isolated bacterial colonies   

Three-phase streaking technique to achieve isolated bacterial colonies. In the first step (1), the inoculation loop is moved across one third of the agar, for the following steps (2 and 3), the loop is changed or sterilized through a flame.

For a quick identification, the following selective agar plates are inoculated parallel to quantitative urine culure (on CLED):

MacConkey agar:

MacConkey is a selective agar for the growth of Gram-negative bacteria. The fermentation of lactose is indicated by the pink color of the colonies [fig. bacterial colonies on MacConkey agar], non-fermenters produce colorless colonies.

figure bacterial colonies on MacConkey agar   

Colonies from gram-negative bacteria on MacConkey agar. The fermentation of lactose is indicated by the red color of the colonies. Public domain figure from Medimicro, http://commons.wikimedia.org.

Columbia-CNA agar:

Columbia-CNA agar is a selective agar for the growth of Gram-positive bacteria (e.g. streptococcus, enteroococcus and staphylococcus). The agar contains the antibiotics colistin and nalidixic acid, which prevent the growth of Gram-negative bacteria. The agar contains intact erythrocytes, the growth of the bacteria may be differentiated according to the ability of hemolysis: α hemolysis (green color of the agar due to oxidation of hemoglobin), β hemolysis (transparent color of the agar due to complete hemolysis) and γ hemolysis (unchanged agar color) [fig. &beta hemolysis on Columbia CNA agar/a>].

figure bacterial colonies on MacConkey agar   

Pronounced β hemolysis by streptococcus on Columbia-CNA agar. With kind permission, Nathan Reading, Halesowen, UK, http://commons.wikimedia.org.

Sabouraud-Dextrose agar:

Sabouraud-Dextrose agar is a selective agar for the growth of fungi like candida and aspergillus.

Approximate identification of bacteria:

Approximate identification of bacteria is possible with the help of the growth on above mentioned selective agar plates and basic biochemical properties:

Approximate differentiation of Gram-negative bacteria after growth on MacConkey agar.
figure Approximate differentiation of Gram-negative bacteria after growth on MacConkey agar

Approximate differentiation of Gram-positive bacteria after growth on Columbia-CNA agar.
(1) Differentiation between Staphylococcus epidermidis and Staphylococcus saprophyticus with the help of novobiocin test on Müller-Hinton agar. Staphylococcus saprophyticus is resistent to novobiocin.
figure Approximate differentiation of Gram-positive bacteria after growth on Columbia-CNA agar

Exact identification of bacteria:

The exact identification is possible with the help of test kits using biochemical properties of the bacteria: e.g. oxidase, catalase, laktase, fermentation, urease, mobility, usage of citrate. Examples of test kits are API (Analytic Profile Index) or VITEK system from the company bioMérieux or the RAS-ID system from the company BIO-RAD.

Antibiotic Sensitivity Test

A defined amount of individual colonies are inoculated on a Müller-Hinton agar plate. Paper plates with different concentrations of antibiotics are placed on the agar plate. The antibiotic will diffuse into the agar and inhibit bacterial growth depending on sensitivity. After incubation of 24 h, the diameter around the paper plates is suggestive for the minimum inhibitory concentration for the tested antibiotic. The minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic which prevents visible growth of a bacterium.


Recommendations for the technique of antibiotic sensitivity tests are published, e.g. in Europe from EUCAST (European Committee on Antimicrobial Susceptibility Testing). Details on the homepage http://www.eucast.org. Several commercial systems simplify the antibiotic sensitivity test, e.g. RAS-ID from the company BIO-RAD or VITEK system from the company bioMérieux.

Index: 1–9 A B C D E F G H I J K L M N O P Q R S T U V W X Y Z


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