HEALTH

Sunday, 5 June 2011

MICROBIOLOGY- URINE CULTURE AND SENSITIVITY


URINE CULTURE AND SENSITIVITY

PRINCIPLE:

The isolation and identification of Significant bacteriuria in urine.  Sensitivity testing should be performed on the organism(s)

Personnel
Student Technician
Assistant Technician
Rotation Technician
Technician
Technologist
Specimen Sorting and numbering
*
Microscopy
*
Culture
*□
*
Interpretation of cultures


Sensitivity test


Reporting


*□
*Under supervision only.

Safety:  Wear gloves

Reception:  Inoculate immediately or refrigerated at 4ºC for not more than 18 hours after collection.

Equipment/Reagent:
Calibrated Standard loop (1/500) or 1/400).
Microscope Slides, Cover glass, Pasteur pipette, Beakers, Racks, Saline, CLED(or BA and Mac) DSTA, (CA) when needed, Peptone water Urea, Citrate, TSI, Antibiotic discs, Plasma, Gram/Z-N Stains when needed, test tubes, Sterile centrifuge tubes and Centrifuge.

DAY 1

Test Procedure:
(Semi-quantitative Standard loop Method)

Mid-Stream Urine (MSU)  is the usual specimen (see others below)
(1)     mix the urine sample carefully by rotation but not by inversion (to avoid any contamination if the lid is loose).
(2)     Dip the standard loop (1/400 or 1/500ml) vertically into the urine and inoculate onto ¼ Cystine Lactose, Electrolyte Deficient (CLED) Agar or Blood Agar (BA) and MacConkey Agar (Mac) plates.  Only the ring of the loop should enter the urine.
(3)     Incubate aerobically at 35-75ºC overnight (For N gonorrhoeae put deposit on Chocolate agar, (send deposit for TB culture to PHL)




                                                                                    Fig 1

Microscopy:
Put 2 drops of well mixed uncentrifuged urine onto a Slide.  Apply a cover slip.  Examine with a x 10, objective for WBC/RBC/Epithelial Cells and bacteria.  To identify type of cells or casts use x 40 objective.  Count number of cells in 10 fields and express the results as per High power field.

In emergency cases
  • Put up direct Sensitivity test on urine containing.
(1)    20 or more WBC per field
(2)    Bacteria
(3)    Both bacteria and 20 or more WBC

DAY 2
(1)    Examine plate.  Note the degree of growth on each ¼ plate (Fig 1)
(2)    Interpretation of culture
i.             No bacterial growth
ii.           No significant growth less than (<) 200 cfu
iii.         Significant growth:-
                  With a 1/400 ml loop approximately 250 cfu
                  With a 1/500 ml loop approximately 200 cfu
This is proportional to > 108 bacteria./Litre (.> 105 bacteria/ml)

(3)    Other considerations
a.       S. typhi – even a few colonies are significant
b.      Suprapubic aspirate) less than 105/ml is significant            Ureteric samples     )
c.       Candida spp – important in diabetics and immunosuppressed patients
d.      *Pseudomonas species….

(4)    Mixed growth
a.       Significant growth of only two types of colonies identify and do sensitivity test.
b.      More than two types of colonies, ask for a repeat specimen




Identification/Sensitivity Tests
Some Common Pathogens

Organism
Gram Result
Test(s)
*  * Sensitivity
E. coli, Klebsiella
Enterobacter
Citrobacter & Other
Coliforms
GNR (LFs)
Indole, Urea Citrate, TSI
Motility Tests or consider
direct API 20E
Ampicillin, Tetracycline Nalidixic Acid,
Nitrofurantoin Pipedidic Acid, Septrin
Gentamicin, Cefuroxime, Augmentin
Pseudomonas spp
GNR (NLFS0
Oxidase, motility
Non-fermenter of Sugars, or
Consider direct API 20E
Gentamicin, Noroxin, Cefotaxime,
Ofloxacin Ceftazidime, Amikacin
Urotractin, ciprofloxacin
Salmonella spp
GNR
(NLFs)
Indole, Urea Citrate TSI,
Motality or Consider direct
Using specific antisera
Chloramphenicol, Cefuroxime
Angmentin, Nalidixic Acid, Noroxin,
Urotractin Gentamicin, Cefuroxime
Nitrofurantoin, Ampicilin
Faecal Streptococcus
(enterococci)
GPC (Chains)
Catalase test, Growth on
MacConkey
As above

**  Alternatively, use available multodisc for Gram negatives or Gram positives

Reporting
                    I. Significant growth
Must be reported with sensitivities as follows

1.      Out patients  - Report oral antibiotics only and keep the Sensitivity of parenteral ones on the request forms.
2.      In-patients – Report both parenteral and oral antibiotics.
List of antibiotics will be amended from time to time, depending on availability.

II.                No significant growth.

III.             Mixed growth
a.       Significant growth of 2 types of organisms – report with sensitivity
b.      More than 2 types of organisms, report as mixed growth.

Repeat culture requested
BY BMS DZAMESI

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