1. Urine culture
D.M.M. Lab.

2. Routine Urine Culture Criteria of specimen rejection 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.

3. *Staphylococcus aureus
Pathogens and commensals
Common pathogens
Commensal flora
Neisseria gonorrhoeae any colony on chocolate or TM agar (special request).
Diphtheroid bacilli
E.coli and other Enterobacteriaceae
Lactobacillus spp
Enterococcus spp
Coagulase negative Staphylococci
*Staphylococcus aureus
Pure culture regardless to the no. of CFUs.
Alpha Haemolytic Streptococci
Staph saprophyticus
Bacillus spp
Corynebacterium jeikeium
Non pathogenic Neisseria spp.
Acinetobacter spp
Anaerobic cocci
Pseudomonas spp
Commensal Mycobacterium
* Gardnerella vaginalis Unusual
Commensal Mycoplasma spp.
Beta -haemolytic streptococci
* Salmonella spp (early stage of infection)
* yeast
Parasites
Schistosoma haematobium
Trichomonas vaginalis
* Diagnostic Microbiology, BAILEY & SCOTT, 9th EDITION

4. 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.

5. 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.
The conditions and concentrations of urine preservatives are defined for 24-hour collections as follows:
Ambient
Room temperature (21°-25° C)
Refrigerated
4° C
Frozen
-20° C
6N HCl
30 mL per 24-hour collection
Acetic Acid 50%
25 mL per 24-hour collection
Na2 CO3
(crystals)
5 g per 24-hour collection
Toluene
30 mL per 24-hour collectioN
6N HNO3
15 mL per 24-hour collection
Boric Acid
10 g per 24-hour collection
Thymol
(10% in isopropanol)
10 mL per 24-hour collection
Suprapubic aspiration

6. Specimen processing

7. 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 refers to urine which contains pus. Defined as the presence of 4 or more neutrophils per high power field.

8. 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.

9. 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).

10. Specimen processing
Culturing

11. 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.

12. Culturing Procedure

13. Colony counting
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.

14. 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.

15. 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.
Most laboratories limit the number of organisms which will be identified when recovered from urine to two.
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.