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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
close this folderUrine
View the documentIntroduction
View the documentSpecimen collection
View the documentCulture and interpretation
View the documentInterpretation of quantitative urine culture results
View the documentIdentification
View the documentSusceptibility tests
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Culture and interpretation

All urine specimens brought to the microbiology laboratory should be examined at once, or placed in a refrigerator at 4 °C until they can be examined. The examination procedure includes the following steps:

1. Examination of a Gram-stained smear.

2. A screening test for significant bacteriuria.

3. A definitive culture for urine specimens found to be positive in the screening test (step 2), and for all specimens obtained by cystoscopy, suprapubic bladder puncture (SBP), or catheterization.

4. Susceptibility tests on clinically significant bacterial isolates.

Preparation and examination of a Gram-stained smear is a necessary part of the laboratory process. Using a sterile Pasteur pipette (one for each sample), place one drop of well mixed, uncentrifuged urine on a slide. Allow the drop to dry without spreading, heat-fix and stain. Examine under an oil-immersion lens for the presence or absence of bacteria, polymorphonuclear leukocytes, and squamous epithelial cells.

One or more bacterial cells per oil-immersion field usually implies that there are 105 or more bacteria per millilitre in the specimen. The presence of one or more leukocytes per oil-immersion field is a further indication of UTI. Non-infected urine samples will usually show few or no bacteria or leukocytes in the entire preparation. In specimens from females, the presence of many squamous epithelial cells, with or without a mixture of bacteria, is strong presumptive evidence that the specimen is contaminated with vaginal flora and a repeat specimen is necessary, regardless of the number of bacteria per oil-immersion field. If results are required urgently, the report of the Gram-stain findings should be sent to the physician with a note that the culture report is to follow.

Screening method

The absence of leukocytes and bacteria in a Gram-stained smear of a clean-catch urine sample prepared as described above is good evidence that the urine is not infected. A urine specimen that is “negative” on careful examination of the Gram-stained smear does not need to be cultured. An alternative simple and effective screening test is the test strip for leukocyte esterase/nitrate reduction. The strip is dipped into the urine specimen as instructed in the package literature. Any pink colour is a positive reaction indicating the presence of leukocyte esterase and/or bacteria in excess of 105 per ml. Urine samples that are positive in the screening test should be cultured as soon as possible to prevent possible overgrowth by nonsignificant bacteria. If the strip does not develop a pink colour it is interpreted as a negative screening test, is so reported, and no culture is indicated. The test strip may not be sensitive enough to detect bacterial counts of less than 105 per ml of urine.

Combined quantitative culture and presumptive identification

A technique that can be recommended for quantitative culture is the filter paper dip strip method of Leigh and Williams,1 used with two different plating media for presumptive identification. The strip method is based on the absorption and subsequent transfer of a fixed amount of urine to a suitable plating agar medium.

 

1 LEIGH, D. A. & WILLIAMS, J. D. Method for the detection of significant bacteriuria in large groups of patients. Journal of clinical pathology, 17: 498-503 (1964).


Fig. 3. Diagram of dip strip (WHO 91125)

The strips are cut from a specific type of blotting paper and measure 7.5 cm long by 0.6 cm wide (see Fig. 3). They are marked at 1.2 cm from one end with a pencil. The strips are made in quantity, placed in a suitable container and autoclaved. Sterile strips are commercially available from Mast Laboratories (Bootle, Merseyside, England) under the name Bacteruritest. A sterile strip is removed from the container for each urine sample to be tested. The marked end is dipped as far as the mark into the thoroughly mixed urine sample. The strip is withdrawn immediately and the excess urine is allowed to be absorbed.

The area below the mark, which will bend over like the foot of an “L”, should then be placed in contact with a plate of brolacin agar1 or purple lactose agar for 2 - 3 seconds. Several strips can be cultured on one plate by dividing the undersurface of the plate into up to 16 rectangles (see Fig. 4). Be sure to identify each rectangular area with the number or name of the patient. Remove a second strip from the container and repeat the procedure exactly, making a second imprint identical to the first. Once the plate is completely inoculated with duplicate impressions, it should be incubated at 35 - 37 °C, and the colonies resulting from each dip-strip imprint counted. With the help of Fig. 4 it is possible to convert the average number of colonies for each pair of dip strips to the number of bacteria per ml of urine.

 

1 Bromothymol-blue lactose cystine agar.

Immediately following the above procedure, inoculate half a plate of MacConkey agar (with crystal violet) with the urine specimen using a sterile loop. The inclusion of a blood agar facilitates the rapid identification of Gram-positive cocci. Plates should be incubated at 35 - 37 °C overnight, and examined on the following day for growth. Identification procedures may then be initiated using well separated colonies of similar appearance. If required, the inoculum for performing the disc - diffusion susceptibility test can be prepared from either of these plates. In this way, the results of both identification and susceptibility tests will be available on the next day.


Fig. 4. Dip-strip impressions on an agar plate, showing conversion from number of colonies to number of bacteria per ml (WHO 91126)

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