MICRO.

CASE STUDY

A 26-year-old woman comes to the traveler's clinic complaining of profound weakness and
severe diarrhea of one and one-half days' duration. She has just returned from Africa where she
worked for two months in a refugee camp on the border of Rwanda and Zaire. On the plane she
developed abdominal bloating, intestinal gurgling and nausea followed by two loose bowel
movements. Soon she was having profuse watery diarrhea occurring hourly. Stools were like
rice-water, clear and without odor.

You meet her in the clinic. She looks very weak but has no fever. Blood pressure is 94/60 lying
down and drops to 72/40 standing. She is admitted to the hospital. Admitting laboratory studies
include the following:

Sodium 138 meq/L

Potassium 2.1 meq/L
Chloride 95 meq/L
CO2 15 meq/L
Fecal leukocytes None seen
Fecal occult blood Negative

QUESTIONS/INSTRUCTIONS (with corresponding points):

1. Make a flowchart in processing the sample starting from receiving until identification.

FLOWCHART OF THE ISOLATION, IDENTIFICATION AND RECOMMENDATION OF Vibrio

cholerae IN A STOOL SAMPLE

STOOL
SAMPLE
Do Gram Staining Gram-negative, comma-shaped rods

And Acid-fast Bacillus Staining AFB-

Inoculate on:
Primary medium: CAP, BAP
Selective and Differential Medium: MC, TCBS with APW
Maintenance media: TSB, NB, NA

Incubate for 18-24 hours at 37°C

Read and describe the colonial morphologies in each medium

Primary medium: CAP (smooth, opaque, iridescent colonies with a
 greenish hue), BAP (initially colonies surrounded by
a zone of greening but later becomes clear due to
hemodigestion)
Selective and Differential Medium: MC (colorless or pale colonies at
first, it becomes reddish or pink due to late
lactose fermentation on prolonged incubation),
TCBS with APW (flat yellow colonies)
Maintenance media: TSB, NB, NA (Turbid broth)

Perform Biochemical Testing:

TSIA, Citrate, Indole, Motility, MR, Nitrate reduction, Oxidase, Urease,
VP, Lactose fermentation, LIA, Ornithine decarboxylase, String test,
Arginine

Incubate for 18-24 hours at 37°C

Read and interpret results:

TSIA: A/A, gas (-), H2S (-)
Citrate: Positive
Indole: Positive
Motility: Positive
MR: Negative
Nitrate reduction: Positive
Oxidase: Positive
Urease: Negative
VP: Variable
Lactose fermentation: Variable
LIA: Lysine deamination (-), Lysine decarboxylation (+)
Ornithine decarboxylase: Positive
String test: Positive
Arginine: Negative

Identification of isolate: Vibrio cholerae

Do Antibiotic Sensitivity/Susceptibility testing in:

Ampicillin, Tetracycline, Nalidixic acid, Chloramphenicol, Amoxicillin,
Augmentin, Ofloxacin, Ciprofloxacin, Doxycycline, Cotrimoxazole,
Gentamicin, Streptomycin, Furazolidone, Azithromycin
2. Why are the petri dish/plates normally incubated bottom-side up?
Petri dishes are normally incubated bottom-side up to minimize risk of contamination and
to prevent the accumulation of water condensation that could cause a colony mix up with each
other that would create problem in counting and proper determination of microbial count. Thus,
with petri dishes being incubated upside down, the rate of evaporation decreases resulting in
proper microbial growth.

3. If you were to choose between Blood Agar and MacConkey Agar for your stool culture,
what would be the best option and why?
For me, I would choose MacConkey Agar because it is a selective and differential
medium that only grows gram-negative bacterial species and further differentiate them based on
their fermentation profile. Additionally, it is used for recovery of fecal bacteria and selects for
non-fastidious gram-negative bacteria. Not to mention that the type of bacteria is already known
during the gram staining which is gram negative, so it fits for this medium which then further
differentiate the gram-negative bacteria based on their different growth characteristics on the
medium to detect the specific organism. In contrast, blood agar is a non-selective medium used
to differentiate bacteria based on their hemolytic properties.

4. What are the identifiers needed in labeling the streak plates?

Petri dishes are labelled around the edge of the bottom of the plate to preserve the area
for observing after it has been incubated. The important identifiers in labelling includes the
plater's name or initials, organism’s name, type of growth medium, and the date.