Advanced Techniques in Diagnostic Microbiology

104 J. Aslanzadeh
Voges–Proskaure (VP) Test
Organisms such as Klebsiella, Enterobacter, and Serratia spp. that utilize the
butylenes glycol fermentation pathway produce acetoin, an intermediate in the
fermentation of butylenes glycol. The VP test detects the production of acetoin by
these organisms. In the presence of air and potassium hydroxide, acetoin is oxidized
to diacetyl, which produces a red-colored complex. The addition of alpha-naphtol
increases the sensitivity of the test.
A 5 mL MR-VP broth tube is inoculated with the organism and incubated at
35 ◦ C for 18–24 h, then 2.5 mL of the broth culture is transferred to a fresh tube and
inoculated with 6 drops of alpha naphtol followed by 3 drops of KOH. A positive
result is indicated by the presence of a red color that develops within 15 min.
No color change indicates negative VP (Koneman et al., 1997; Murray et al.,
2003).
Pseudosel Agar Slant
Pseudosel agar is a medium used for the identification of Pseudomonas aeruginosa.
Magnesium chloride and potassium sulfate in the medium enhance the
production of pyocyanin, a blue-green, water-soluble, nonfluorescent phenazine
pigment. P. aeruginosa is the only Gram-negative rod known to excrete pyocyanin.
In addition to the promotion of pyocyanin production, pseudosel agar also enables
the detection of fluorescent products by some Pseudomonas species other than P.
aeruginosa. Streak the surface of the pseudosel agar slant, and incubate at 35 ◦ C
in non-CO 2 for 18–24 h. A blue-green pigmentation surrounding the growth on
the agar slant indicates a positive reaction. No pigmentation indicates a negative
reaction.
Negative pseudosel slants should be examined under short wavelength (254 nm)
ultraviolet light to check for fluorescent products produced by some Pseudomonas
species. Pseudomonas aeruginosa typically produces fluorescein as well as
pyocyanin (BD Microbiology Systems, 1992).
Urea Agar Slant
Microorganisms that possess the enzyme urease are capable of hydrolyzing urea,
which releases ammonia. This reaction raises the pH of the medium and is detected
by phenol red, which turns pink-red above pH 8.0. The color change first appears in
the slant because the oxidative decarboxylation of amino acids in the air-exposed
portion of the medium enhances the alkaline reaction. The color change eventually
spreads deeper into the medium.
Streak the surface of the urea agar slant with a heavy inoculum of a pure culture.
Incubate at 35 ◦ C in non-CO 2 for 18 to 24 h. Production of intense pink-red color
on the slant, which may penetrate into the butt, is considered a positive reaction.
No color change indicates negative a reaction.
The medium is not specific for urease. The utilization of peptones or other
proteins in the medium by some urease-negative organisms may raise the pH due