Urine culture is performed in order to specifically identify organisms that may be causing a urinary tract infection (UTI). Urine in the bladder is often sterile as it is free from microorganisms. It becomes inundated with microorganisms (especially those of the micro-flora) when it leaves the bladder. UTI is very common in patients that are under catheterization and those having a poor personal hygiene. The source of urine sample for urine culture analysis can be mid-stream urine (MSU) or clean catch urine from a patient using a catheter (i.e. in critically ill patients who cannot naturally pass out urine).
AIM: To isolate and identify pathogenic bacteria from urine specimens of patient’s as an aid in the diagnosis of urinary tract infections.
MATERIAL/APPARATUS: Mid – stream urine (MSU) specimen, inoculating loop, Bunsen burner, incubator, cystein lactose – electrolyte deficient (CLED) agar, blood agar (BA), grease pencil.
- Label the relevant agar plate (e.g. CLED and BA) with the patient’s name and laboratory number using a grease pencil.
- Mix the urine specimen by rotating the container.
- Using a sterilized inoculating loop, collect a loopful of the specimen and inoculate it on the culture media plates (CLED & BA).
- Incubate both plates in the incubator at 35oC overnight.
- Examine both plates for significant bacterial colonies.
- Perform biochemical testing (Citrate test and Indole test) and Gram staining.
- Perform antimicrobial susceptibility testing only when a pathogen has been isolated.
NOTE: It is not necessary to culture urine specimens that are microscopically and biochemically normal. Culture is only required when the urine specimen contains bacteria (as indicated by Gram smear), cells, casts, nitrite, or protein.
REPORTING OF THE RESULT:
Report bacterial numbers by counting the approximate number of colonies, and estimating the number of bacteria by colony forming unit per ml (CFU/ml). For bacterial counts, the following applies:
- Bacterial counts less than 104/ml = Not significant.
- Bacterial counts between 104 – 105/ml = Doubtful significance.
- Bacterial numbers more than 105/ml = Significant bacteriuria.
A detection threshold of 105 CFU/ml of bacterial colonies resulting from a urine culture plate is significant of a bacteriuria.
Look particularly for E. coli, Klebsiella, Pseudomonas aeruginosa and Staphylococcus aureus e.t.c on the CLED plate. S. aureus cause haemolysis on BA, and this feature can be used to distinguish it from other bacteria. Other pathogenic microorganisms found in urine include Candida species, Streptococcus pneumoniae, Proteus mirabilis, Enterococcus species and Listeria monocytogenes.
BA is used to isolate fastidious organisms e.g. Staphylococcus and Streptococcus that require additional nutrient like blood to grow.
CLED is a non – inhibitory differential medium which is specially used to isolate urinary pathogens from urine specimens. It allows the growth of both Gram positive and Gram negative pathogens. Lactose fermenting organisms appear yellow on CLED because the agar is bromothymol blue. CLED is electrolyte – deficient, and this prevents the growth of Swarming organisms like Proteus species.