Case study 1: Gram positive cocci

You are working as a medical laboratory technologist (MLT) in the microbiology laboratory.
One day you have received a urine specimen with a request form stated the clinical history
of the patient. The patient is a female, 18 year old with having difficulty to urinate.
Sometimes she felt burning sensation while urinate and incontinence. The doctor who
checked the patient request tests for urine FEME and urine for culture and sensitivity. You
have done the tests. There was a bacteria present in the urine FEME test. The results you
get for the species identification is streptococcus pyogenes that resistant towards
ciprofloxacin.

1. Briefly explain the procedures of urine FEME and urine culture.

Urine FEME
a. Collect a mid clean fresh urine sample in a clean sterile urine container and labelled.
b. Check for colour and odor by naked eyes. Odorless and colourless.
c. Dip strip into the urine and remove excess urine. Left the strip to stand for 1 to 2
minutes in room temperature.
d. Read the change of colour for each parameter by comparing with the colour chart
manually using naked eyes.
e. Read the strip automatically using automated reagent strip reader.
f. Spin the sample in a centrifuge at around 1500rpm for 3 to 5 minutes.
g. Discard the supernatant at the bottom of the test tube.
h. Gently flick the bottom of the tube the sendimetion at the bottom of the tube in the
small volume of urine. Use dropper to place one drop of the sendimention onto slide.
i. Put the slide under the microscope and record the result.
j. UTI’s positive occur white blood cell, red blood cells, nitrate positive, bacteria. Odorless,
colourless = negative.

Urine Culture
a. Collect the fresh urine sample in a sterile urine container and labelled.
b. Obtain culture and sensitivity first before all test. Do not contaminate sample.
c. Fill the urine into the test tube and mix times to dissolve preservative and labelled the
test tube.
d. Use Mac Conkey’s agar or blood agar and labelled name and date.
e. The inoculating wire loop heated use bunsen burner until it is red hot and the allows it
to cool.
f. Use inoculation wire loop and dip into well mixed-urine. Quickly make a single streak
down on the media with the loop containing urine.
g. Immediately streak the inoculating loop very gently over a quarter of the plate using a
back and forth motion. Streak back and forth across the plate perpendicular to the
original inoculum with the same calibrated loop and same media plate.
h. Heated the loop again and allow it to cool. Going back to the edge of area 1 that
streaked, extend the streaks into the second quarter of the plate.
i. Procedure 4 repeated into the third quarter of the plate (area C)
j. Procedure 4 repeated again but this time extend the streaks into the center fourth of
the plate (area D)
k. The plate incubated in the incubator at 37oC for 24 hours.
l. The colony morphology grows observed after 24 hours incubation.

2. What is the antibiotic of choice to treat streptococcus pyogenes instead of

ciprofloxacin.
Amoxicillin. Penicillin. Bacitracin. Cephalexin. Ceftriaxone.

3. Examine the colonial morphology of streptococcus pyogenes on blood agar plate and
describe the cultural characteristics based on the following:

Streptococcus pyogenes on blood agar

Bacteria Size Margin Elevation Texture Pigment Type of
hemolysis
Strep. >0.5mm Entire Flat Shinny Smooth Beta-hemolysis
pyogene
s

4. Microscopic observation after gram staining.

Bacteria Gram Stain Shape Arrangement
Strep. pyogenes Positive Cocci Chain.
5. What are the biochemical tests used to identify gram positive cocci bacteria.

Gram positive cocci

Catalase test

Negative Streptococus
species

Growth on blood agar

Beta hemolytic growth in

presence in bacitracin

No, sensitive. Yes, resistant.

Streptococcus pyogene Another beta-hemolytic
streptococcus

Pyrrolidonyl (PYR Test)

Positive: agglutination

Lancefield group. Used

serum of patient.

Negative: no agglutination
REFERENCES
 https://www.slideshare.net/doctorrao/culturing-of-urine-skill-based-learning
 https://www.gundersenhealth.org/app/files/public/6784/Lab-Policies-RegionalAffiliate-
Urine-Culture-Lab-8842.pdf
 https://slideplayer.com/slide/4375319/
 Microbiology Lectures Notes