URINE CULTURE AND INTERPRETATION

1. URINE SPECIMEN
GROUP 03

2. Why urine collection.
It's used to detect and manage a wide range of disorders, such as urinary tract
infections, kidney disease and diabetes.
In microbiology we deal with microorganisms causing infections in urine.
Possible ones examined in urine are;
1. Mostly gram negative bacteria - Enterobacteriaceae
Escherichia coli
Proteus species
Pseudomonas aeruginosa
Klebsiella strains
Salmonella Typhi
Salmonella Para-typhi
Neisseria gonorrhoeae

3. 2. Gram positive bacteria
Staphylococcus saprophyticus
Hemolytic streptococci
3. Protozoans
T. vaginalis, Schistosoma haematobium, micofilaria.
4. Fungi species
Candida species but may also be caused by Cryptococcus neoformans,
Aspergillus species, and the endemic mycoses.

4. Urine collecting methods.
sterile urine bag, urethral catheterization (CATH), suprapubic aspiration (SPA),
or clean-catch (CC), dry sterile leak proof container.

5. Types of urine Specimen
1. First Morning Specimen - a specimen obtained during the first
Best for:
• Nitrite
• Protein
• Microscopic examination
2. Postprandial – is a specimen obtained 2 hours after meal.
Good for glucose level examination.
3. 24- Hour specimen - a specimen obtained within 24 hours.
• determination of protein levels quantitatively.

6. 4. Mid- stream Specimen - a specimen obtained from the middle part
of the first urine.
• It is commonly used for routine urinalysis.
• It is also important for bacteriological urine culture.
Midstream urine (MSU) for microbiological examination is collected as follows:
1. Give the patient a sterile, dry, wide-necked, leak-proof container and request a 10–20 ml
specimen.
Female patients: Wash the hands. Cleanse the area around the urethral opening with
clean water, dry the area with a sterile gauze pad, and collect the urine with the labia held
apart.
Male patients: Wash the hands before collecting a specimen (middle of the urine flow).
2 Label the container with the date, the name and number of the patient, and the time of
collection.
As soon as possible, deliver the specimen with a request form to the laboratory. When
immediate delivery to the laboratory is not possible, refrigerate the urine at 4–6 °C. When
a delay in delivery of more than 2 hours is antici- pated, add boric acid preservative to the
urine.

7. Specimen processing
Culturing
CLED Agar

8. Mix the urine sample to re-suspend microorganism present.
Dip a 1 μl calibrated loop in vertical position in the urine and
remove the loop and use the collected fluid to inoculate CLED,
Blood and MacConkey agars respectively.
Culturing Procedure

9. Culturing Procedure

10. .
•Contains lactose as substrate and Neutral red as an indicator
•Bacteria fermenting lactose produce acid and this changes
the colour of the indicator and the colonies turn pink
Used: To differentiate lactose fermenters (E. coli, Klebsiella)
from non-lactose fermenters (Salmonella, Shigella)
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1. Mac Conkey’s agar

11. Results of Lactose Fermentation on MacConkey
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12. 2. Blood agar
• Used to differentiate hemolytic and non hemolytic bacteria
• Most gram negative bacteria show no hemolysis on blood
agar which are mostly found in urine specimen i.e klebsiella,
e-coli and proteus ssp.
klebsiella
E-coli
proteus

14. Cystine lactose electrolyte-deficient (CLED) agar is
widely used by laboratories to isolate urinary
pathogens because:
 It gives consistent results and allows the growth of
both Gram negative and Gram positive pathogens.
The indicator in CLED agar is bromothymol blue and
therefore lactose fermenting colonies appear yellow.
The medium is electrolyte-deficient to prevent the
swarming of Proteus species.

15. 1. E-coli
are lactose fermenters on CLED yellowish colonies and reddish/pinkish colonies in MCA
Biochemical test
a. indole test – using Kovac reagent if red ring is formed its indole positive
2. Klebsiella
Are lactose fermenters large mucoid colonies
Biochemical test
a. Citrate test – if it turns blue its citrate positive
b. Urea test – if it turns pinkish its positive
3. Proteus mirabilis
On blood agar it swarms but not in CLED (PREFERED MEDIA), its non lactose fermenter
Biochemical test;
a. Urea test – its positive pinkish
4. Pseudomonas aeruginosa
Causative agent of UTI, its non lactose fermenter
Biochemical test.
a. Oxidase test = its positive

16.  A plate count of 100,000 CFU/ml of pure culture should be considered positive
and isolated organism should be identified and sensitivity test will be performed.
 A plate count between 10,000 – 100,000 CFU/ml is considered suspected .
A plate count less than 10,000 CFU/ml is considered negative.
ml)
in
plated,
(volume
(dilution)
counted
colonies
#
sample
original
in
ml
CFU

Colony counting

18. Count the approximate number of colonies. Estimate the
number of bacteria, i.e. colony-forming units (CFU) per ml of
urine. Report the bacterial count as:
● Less than 10 000 organisms/ml (104/ml), not significant.
● 10 000–100 000/ml (104–105/ml), doubtful significance
(suggest repeat specimen)
● More than 100 000/ml (105/ml), significant bacteriuria.
Interpretation of quantitative urine culture result.

20. EXAMPLE
If 25 E. coli colonies are counted and a 1/1000 ml loop was
used, the approximate number of CFU per ml of urine: 1000 X
25 = 25 000, Such a count would be reported as: 10 000 –
100,000 E. coli/ml
NOTE:
It is generally agreed that if more than 10 per 5 colonies/mL
are cultivated from a properly collected and properly cultured
urine specimen, this constitutes strong evidence of active
urinary tract infection.

21. Sensitivity tests.
We use MHA in performing sensitivity tests using four to five antibiotic drugs.
Drugs for gram negative rods are;
1. Tetracycline
2. Contrimazole
3. Ciproflaxone
4. Gentamycin
5. Erythromycin
6. Penicilin
Incubate over night and look for zone of inhibition. We report in mm in
diameter.