LABORATORY

Question 1

Pseudomonas aeruginosa: Specimens

Answer 1

• Pus
• Sputum
• Urine
• Blood
• Cerebrospinal fluid
• Other material as indicated by the type of infection

Question 2

Gram-negative bacilli

No specific morphologic characteristics differentiate [?] in specimens from enteric or other Gram-negative rods.

Answer 2

pseudomonads

Question 3

P. aeruginosa grow well on routine laboratory media such as

Answer 3

5% sheep BAM, and MAC or EMB

Question 4

-35-37°C; growth at [?] helps differentiate it from other Pseudomonas species that produce fluorescent pigments, i.e., P. fluorescens, and P. putida)

• [?]or ambient air.
• [?] hours

Answer 4

42°C

CO2

24 to 48

Question 5

Colonies are large with a spreading periphery and are often β-hemolytic.

Answer 5

Pseudomonas aeruginosa: On BAM

Question 6

Growth appear to have a metallic sheen and a
scaling appearance sometimes described as
alligator skin morphology.

Answer 6

Pseudomonas aeruginosa: On BAM

Question 7

Colonies typically produce a sweet “grape-like” or

“corn tortilla-like” odor.

Answer 7

Pseudomonas aeruginosa: On BAM

Question 8

Can produce multiple colony types: may be

mucoid, or rough.

Answer 8

Pseudomonas aeruginosa: On BAM

Question 9

P. aeruginosa imparts a blue-green color to the medium. This is produced by combination of two water-soluble and diffusible pigments:

Answer 9

pyocyanin (blue) and pyoverdin (yellow/yellow-green)

Question 10

An uninoculated tube (A) is shown for comparison. Note the blue-green color (B) that diffuses into the medium.

Answer 10

P. aeruginosa on tryptic soy agar

Question 11

Disk diffusion antimicrobial susceptibility test of P. aeruginosa on [?]. Note the blue-green pigments.

Answer 11

Mueller-Hinton agar

Question 12

is a fluorescein pigment that fluoresces white to blue-green under long wavelength (400-nm) ultraviolet light source (e.g., Wood’s lamp)

Answer 12

Pyoverdin

Question 13

a yellow-green pigment which is water soluble and fluoresces white to blue-green under longwavelength (400-nm) ultraviolet light.

Answer 13

pyoverdin

Question 14

produces pyocyanin

Answer 14

P. aeruginos

Question 15

is a fluorescein pigment that fluoresces white to blue-green under long wavelength (400-nm) ultraviolet light source (e.g., Wood’s lamp)

Answer 15

pyoverdin

Question 16

Production of pyoverdin is particularly enhanced in media with a high phosphate concentration such as [?]. [?] are also suitable for demonstrating fluorescence.

Answer 16

Flo medium

King’s medium B, Sellers’ medium, and Mueller-Hinton agar

Question 17

Fluorescence may be enhanced if cultures are incubated at [?] rather than at 35°C to 37°C.

Answer 17

20°C to 30°C

Question 18

water-soluble, diffusible, blue pigment

Answer 18

Pyocyanin

Question 19

water-soluble, diffusible, green (or yellow-green) pigment

Answer 19

Pyoverdin

Question 20

red pigment

Answer 20

pyorubin

Question 21

black pigment; brown to black

Answer 21

pyomelanin

Question 22

• Non-lactose fermenting (NLF) colonies.
• May show green pigmentation, or metallic sheen.
• May be mucoid.

Answer 22

Pseudomonas aeruginosa: On MAC

Question 23

[?] demonstrating growth of a mucoid variety of P. aeruginosa typical of the strains isolated from the sputum of patients with cystic fibrosis. Note that in the heaviest growth area, there appears to be some pyocyanin pigment developing.

Answer 23

MAC

Question 24

To distinguish P. aeruginosa from the gram-negative bacilli of the family Enterobacteriaceae

Answer 24

Oxidase (+)

Question 25

No acid production in indicates the inability of nonfermenting P. aeruginosa to utilize the lactose, glucose, or sucrose in TSI, or the lactose or glucose in KIA.

Answer 25

TSI or KIA: K/K

Question 26

IMViC:

Answer 26

• • • +

Question 27

Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively.

The end products of fermentation are relatively strong mixed acids that can be detected in a conventional fermentation test medium.

However, the acids formed in oxidative degradation of glucose are extremely weak, and the more sensitive oxidation-fermentation medium of Hugh and Leifson (OF medium) is required for their detection.

Answer 27

Oxidation-Fermentation (OF) Test

Question 28

Oxidation-Fermentation (OF) Test: Medium

Answer 28

Hugh–Leifson OF medium

Question 29

The OF medium of Hugh and Leifson differs from carbohydrate fermentation media as follows:
• The concentration of peptone is [?]
• The concentration of carbohydrate is [?]
• The concentration of agar is [?]making it semisolid.

Answer 29

decreased from 1% to 0.2%.

increased from 0.5% to 1.0%.

decreased from 1.5% to 0.3%,

Question 30

The [?] reduces the formation of alkaline amines that can neutralize the small quantities of weak acids that may form from oxidative metabolism.

Answer 30

lower protein/carbohydrate ratio

Question 31

The relatively [?] serves to increase the amount of acid that can potentially be formed.

Answer 31

larger amount of carbohydrate

Question 32

The [?] of the agar permits acids that form on the surface of the agar to permeate throughout the medium, making interpretation of the pH shift of the indicator easier to visualize.

Answer 32

semisolid consistency

Question 33

[?] can also be observed in this medium.

Answer 33

Motility

Question 34

Note that two tubes of each carbohydrate medium
are required for the test.
i. Inoculate each tube with the unknown organism, using a straight needle, stabbing the medium three to four times halfway to the bottom of the tube.
ii. To one tube of each pair, cover with a 1-cm layer of sterile mineral oil or melted paraffin. (CLOSED TUBE).
iii. Leave the other tube open to the air. (OPEN TUBE).
iv. Incubate both tubes at 35°C and examine daily for several days.

Answer 34

Oxidation-Fermentation (OF) Test

Question 35

Oxidation-Fermentation (OF) Test: Result

A change in the initial green color of the medium to a yellow color indicates [?] from utilization of glucose.

Answer 35

acidification

Question 36

produce acid only in the open tube exposed to atmospheric oxygen.

Answer 36

Oxidizers

Question 37

produce acid in both tubes.

Answer 37

Fermenters

Question 38

bacteria are inert in this medium, which remains at an alkaline pH after incubation.

Answer 38

Nonsaccharolytic

Question 39

Acid (yellow) is seen only in the top portion of the open tube, indicating that the organism is capable of oxidizing glucose but incapable of fermenting glucose.

Answer 39

OXIDATIVE UTILIZATION OF GLUCOSE by P. aeruginosa in Hugh-Leifson OF medium.