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