Lecture 5 Flashcards
Quantitative streaking
FOR URINE for a UTI sample Plates inoculated for quantitation are usually streaked with a 1:100(0.01mL or 10µL) or 1:1000(0.001mL or 1 µL) loop Plated using a calibrated loop Cfu/ml
isolated colonies are counted and then the number is calculated as:
the # of colonies x 1000 (if a 0.001mL loop was used) = the no.of CFU/ml of sample.
If a 1:100 loop is used then the count is multiplied by 100.
In a urine specimen if > 100,000 CFU/ml of urine sample it indicates UTI,
Normal urine that is simply contaminated with normal flora a count of 100-1000/mL is considered normal.
Further biochemical test can be done to identify the bacteria
Urine Cultures
Cultures are done to diagnose bacterial infections of the urinary tract:
Bladder,ureter,kidney and urethra
UTI are of two main types
1- Lower Urinary tract- Cystitis-infection of the bladder
2-upper urinary tract –Pyelonepritis-infection of the kidney
Preferred Urine Sample
The commonly used sample is the clean-catch midstream urine sample
Should be collected in a STERILE CONTAINTER
Must be ideally plated within 30 MINS and if not should be refrigerated and plated within 2 hrs.
Patient should be given proper instructions for collecting the sample.
Sample should be collected before the start of antibiotics ,or If on ,Person should be off antibiotics for several days prior to collection
Patient Instructions for Urine Collection
- Explain procedure to patient.
- Ensure patient privacy
- Advice patient to wash their hands pre-procedure
- Ensure patient cleans urethral meatus with 0.9% sodium chloride soaked cotton wool balls or gauze in a downwards motion front to back.
- Instruct patient to collect midstream specimen in sterile container after passing a small amount of urine into the toilet and then finish passing urine into the toilet
- Label specimen correctly with patient’s name, medical record number and time and date of collection
7.Ensure the lid is tightened on container. If open discard and recollect
Note on the request form if the patient is menstruating. - Place specimen container and request form in a biohazard specimen bag for transportation to the laboratory or store refrigerated for collection
The two procedures performed as part of the urine culture examination are
- colony count and
- Antibiotic susceptibility,
So that is why it is known as a C/S test (Culture on proper media and Sensitivity – do antibiotic panel based on what is grown)
Normal urine that is simply contaminated with Normal flora a count of 100-1000 cfu/ mL is considered normal. 1 colony is 1 cfu
To distinguish contamination of urine by normal flora from UTI,it is imp. and necessary to determine the number of organism present /mL of specimen
Current guidelines for infection
> 10^5 CFU/mL indicates infection
<10^4 CFU/mL indicates urethral or vaginal contamination
10^4 and 10^5 CFU/mL needs to be evaluated based on clinical information.
An increased number of WBC-
PMNs in a urine sediment along with the result of urine culture, increases the diagnostic value of both the tests
In regards to the above scenario, Which tests on dipstick would be positive
Turbid, sediment, nitrite and leukocyte esterase would come back positive for infection
Streaking the plate for colony count
The commonly responsible org for UTI is E.coli ,a gram-ve rod that is also the normal flora of the intestine. 90% of the time
Other gram-ve bacilli such as Pseudomonas,Proteus and Klebsiella can also cause UTI.
Gram +ve bacteria such as Staphylococcus aureus, Staphylococcus saparophyticus – tampon sickness and Streptococcus also cause UTI
urine specimen are streaked onto primary media like
Blood agar ,EMB or MAC and now on the CLED medium - UTI
Blood agar being an enriched medium, most UTI pathogens(both Gram +ve and Gram-ve) grow on Blood agar
EMB and MAC inhibit Gram +ve orgs and indicate the fermentation reactions of Gram-ve orgs like E.coli
CLED medium
Cystine-Lactose- Electrolyte-Deficient agar
CLED Agar is a differential culture medium for use in enumerating bacteria in urine. It supports the growth of urinary pathogens and contaminants but prevents undue swarming growth of Proteus species due to its lack of electrolytes.
No electrolytes prevents proteus growth
EMB Medium-
same as MAC look up indicators
This is another medium which is used for the gram negative bacteria.
Intense lactose eosin precipitates gram neg . Very shiny
It is differential because of its components; sugar lactose, sucrose, and dyes Eosin Y and methylene blue.
growth is colorless, off-white or a very light and dull pink, the pH has largely been unaffected: is probably a Gram negative noncoliform.
If good growth that is pink, sugar utilization has slightly lowered the pH: is probably a Gram negative coliform.
If good growth that is purplish or has a purple center, sugar utilization has lowered the pH: is probably a Gram negative coliform.
If good growth with a greenish metallic sheen, strong sugar utilization has greatly lowered the pH: is probably a Gram negative coliform.
(If poor or no growth, recall it is probably a Gram positive organism.)
Streaking a urine culture plate
- The appropriate medium is taken.
- The sample is well mixed
- A vertical streak is made down the center of the plate using a calibrated loop.
- Several streaks @ right angle to the initial streak are made, crossing over the original streak several times.
Plates that show no growth may be discarded and reported as No growth
Growth on both – gram Neg
Growth on blood but not MAC – Gram positive
Colony count and interpretation
The plates with growth are observed for
1.the types of colonies ,
distinguishing characteristics like hemolysis on the BAP
the number of colonies
Gram stain is done of the colonies from BAP.
Quality assessment for Urine Culture
The specimen for culture must be collected before antibiotic therapy has begun
Contamination of urine should be avoided
Since a bacterial UTI is caused by only one type of bacteria, presence of two or more type of colonies on primary plate suggests, the urine was not clean-catch
If routine urinalysis is ordered, the culture should be setup first
Only calibrated loops should be used.
To ensure media sterility, one plate of each type should be incubated at 35oC
