FLOWCHART OF THE ISOLATION, IDENTIFICATION AND RECOMMENDATION OF

Pseudomonas aeruginosa IN A BLOOD SAMPLE

BLOOD
SAMPLE
Do Gram Staining Gram-negative rods

And Acid-fast Bacillus Staining AFB-

Inoculate on:
Primary medium: BAP
Selective and Differential Medium: MC, Cetrimide agar
Maintenance media: TSB, NA, NB

Incubate for 18-24 hours at 37°C

Read and describe the colonial morphologies in each medium

Primary medium: BAP (typical metallic sheen, beta-hemolytic)
Selective and Differential Medium: MAC (flat, smooth, colorless, non-
lactose fermenting colonies),
Cetrimide agar (pigment production)
Maintenance media: TSB, NA, NB (Turbid broth)

Perform Biochemical Testing:

TSIA, Oxidase test, Catalase test, Indole test, MR, VP, Citrate, Urease,
Motility, Coagulase, Arginine dihydrolase (ADH), Lactose fermentation,
OF test, Gluconate Production

Incubate for 18-24 hours at 37°C

Read and interpret results:

TSIA: K/K, gas (-), H2S (-)
Oxidase: Positive
Catalase: Positive
Indole: Negative
MR: Negative
VP: Negative
Citrate: Positive
Urease: Negative
 Motility: Positive
Coagulase: Negative
ADH: Positive
Lactose fermentation: Negative
OF test: Positive
Gluconate Production: Positive

Identification of isolate: Pseudomonas aeruginosa

Do Antibiotic Sensitivity/Susceptibility testing in:

Cephalosporins, Carbenicillin, Colistin, Gentamicin, Polymyxin,
Quinolones, Streptomycin, Ceftriaxone, Ampicillin, Erythromycin,
Chloramphenicol, Amikacin

Answer the questions briefly.

Question 1:
What are the virulence factors of P. aeruginosa and its mode of transmission?
Pseudomonas aeruginosa is an opportunistic pathogen to humans and its pathogenicity
may be linked to the production of virulence factors. The main virulence factor of these bacteria
is biofilm formation. In addition, other principal virulence factors include elastase, phospholipase
C, protease A, exotoxins and cytotoxins, flagella and pili, pigment production, and QS regulatory
system proteins, which regulate both virulence factor transcription and biofilm formation. They
are mostly found in moist environment like soil and water. Regarding its transmission, it can be
spread directly from human to human such as in contact with dirty hands. It can also be
transmitted indirectly through contaminated water, soil, objects, equipment and surfaces.

Question 2:
In preparation of blood agar (BA) what is the preferred blood used and why? And what is
the most striking feature of these bacteria?
Sheep blood is the preferred blood used since sheep RBCs are most sensitive to the
hemolytic toxins released by bacterial cells resulting to hemolytic zones around the colonies
over the period of time which describes the hemolytic pattern of the bacteria. The most striking
feature of these bacteria is their ability to produce a variety of colorful water soluble pigments
such as pyocyanin (blue) which refers specifically to its “blue pus” attribute, pyoverdin (yellow-
green to yellow-brown and fluorescent), pyomelanin (black), and pyorubin (red), and for its
characteristic “corn taco” or “grape”-like odor.