Transcript Lab7 Urine

‫بسم هللا الرحمن الرحيم‬
2013-2014
L/O/G/O
Diagnostic Medical Microbiology-Laboratory Manual
Routine Urine Culture
Aim of the test :
An etiological diagnosis of bacterial urinary tract infection by
semi quantitative cultivation of the urine with identification
and susceptibility test of the isolated bacteria(s).
Types of specimen:
Urine (Midstream urine), suprapubic aspiration, catheterized
urine.
Note: First morning specimens yield highest bacterial counts
from overnight incubation in the bladder, and are the best
specimens.
Criteria of specimen rejection :
Un-refrigerated specimen older than 2 hours may be subject
to overgrowth and may not yield valid results; unlabeled
specimen; mislabeled specimen; specimen in expired
transport container; 24 hours urine specimens.
Pathogens and commensals :
Common pathogens
Neisseria gonorrhoeae any colony on chocolate or
TM agar (special request).
E.coli and other Enterobacteriaceae
Enterococcus spp
*Staphylococcus aureus
Commensal flora
Diphtheroid bacilli
Lactobacillus spp
Coagulase negative Staphylococci
Alpha Haemolytic Streptococci
Pure culture regardless to the no. of CFUs.
Staph saprophyticus
Corynebacterium jeikeium
Acinetobacter spp
Pseudomonas spp
Bacillus spp
Non pathogenic Neisseria spp.
Anaerobic cocci
Commensal Mycobacterium
* Gardnerella vaginalis Unusual
Commensal Mycoplasma spp.
Beta -haemolytic streptococci
* Salmonella spp (early stage of infection)
Parasites
Schistosoma haematobium
Trichomonas vaginalis
* yeast
Pre specimen processing
Patient preparing:
Instruct the procedures for the patient Specimen collection
Collection of midstream urine for bacterial investigation:
Patient not needing assistance:
Give the patient a suitable container.
Instruct the patient before the collection, preferably with illustration.
Tell the patient not to touch the inside or rim of the container.
Pre specimen processing
Who will collect the specimen:
 Midstream urine is collected by the patient.
 If disabled, nursing staff will assist in collection.
 For catheterized specimen, nursing staff will collect the specimen.
 Suprapubic aspiration is performed by the physician.
Quantity of specimen :
To fill line in transport tube (~20 mL).
Time relapse before processing the sample :
The maximum time allowed for processing a urine
sample is 2 hours from the time of collection.
Storage :
At room temperature unless delay is inevitable; it
must be refrigerate or mixed with preservative like
boric acid.
Suprapubic aspiration
Specimen processing
Initial report
The use of dipstick designed to detect the presence of urine nitrite and
to indirectly estimate the number of segmented neutrophiles through the
detection of leukocyte esterase activity. Rationale for the nitrate test is
that most urinary tract infections are caused by nitrate reducing members
of the family Enterobacteriaceae.
Leukocyte esterase (LE) is produced by segmented nutrophiles; this
test when performed alone correlated with ten or more white blood cells
per high power field in the urine with a sensitivity in the range 88% and
specificity 94%.
 Pyuria: the increased number of WBC in urine sample.
 sterile pyuria: is a condition arises when there is an
elevated in WBC in urine and negative culture.
Screening test
As many as 60% to 80% of all urine specimens received for culture by
the acute care medical Center laboratory may contain no etiological agents
of infection. Procedure developed to identify quickly those urine specimens
that will be negative on culture, thus to circumvent excessive use of media,
technologist time, and the overnight incubation period. The gram stain is
the easiest, least expensive, and probably the most sensitive and reliable
screening method for identifying urine specimens that contain greater than
10^5 CFU/ml.
A drop of well-mixed urine is allowed to air dry.
The smear is stained and examined under oil immersion (1000x).
Presence of at least one organism per oil immersion field.
( examining 20 fields ) corelates with significant bacteriuria (>10^5 CFU/ml).
Specimen processing
Culturing:
Culturing Procedure:
Mix the urine sample to re-suspend microorganism present.
Dip a 1 μl or 10 μl calibrated loop in vertical position in the urine and
remove the loop and use the collected fluid to inoculate Nutrient, Blood
and MacConkey agars respectively.
Culturing Procedure:
Colony counting
CFU
# colonies counted
in original sample 
ml
(dilution) (volume plated, in ml)
 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.
Colony counting
Catheterized urine:
Plate count > =1000 CFU/ml
Suprapubic urine :
Any colony is significant
Routine urine culture(voided urine specimen):
Plate count >=105 CFU/ml
Post specimen processing
Interfering factors:
 Patient on antibiotic therapy.
 Improper sample collection.
Result reporting:
 Report wet mount as an initial report.
 Report the isolated pathogen and its sensitivity pattern as a final
report.
Turn around time:
 Wet mount results should be available 1 hour after specimen
receipt.
 Isolation of a possible pathogen can be expected after 2-3 days.
 Negative culture will be reported out 1-2 days after the receipt of
the specimen.
Additional information
 A single culture is about 80% accurate in the female; two containing
the same organism with count of 10^5 or more represents 95%
chance of true bacteriuria; three such specimens mean virtual certainty
of true bacteriuria.
 Single clean voided specimen from an adult male may be
considered diagnostic with proper preparation and care in specimen
collection.
 Urinary tract infection is significantly higher in women who use
diaphragm-spermicide contraception, perhaps secondary to increased
vaginal pH and a higher frequency of vaginal colonization with E. coli.
 If the patient is receiving antimicrobial therapy at the time the
specimen is collected, any level of bacteriuria may be significant.
 Failure to recover aerobic organisms from patients with pyuria or
positive Gram's stains of urinary sediment may indicate the presence
of mycobacteria or anaerobes.