Advanced Techniques in Diagnostic Microbiology

92 J. Aslanzadeh
tryptophane resulting in the production of indole. This test can be performed on
organisms grown on a BAP after 24 h incubation.
Filter paper is placed in a Petri plate and saturated with 3–4 drops of 1% solution
of p-dimethylaminocinn-amaldehyde. Isolated colony(ies) from a 24 h culture
grown on a BAP is rubbed into the filter paper with a wooden applicator stick
or inoculating loop. Appearance of a blue color immediately or within 30 s of
inoculation indicates a positive reaction; no blue color within 30 s indicates a
negative reaction.
The test must be performed from BAP. False-negative results will occur from
MacConkey agar and TSI slants because there is not a sufficient source of tryptophane
in these media, and false positives will occur if indole-positive organisms
are present in mixed cultures (Forbes et al., 2002).
Slide Coagulase Test
Coagulase is a thermostable enzyme found primarily in S. aureus and is used to
differentiate S. aureus from other commonly isolated staphylococci. Two forms
of coagulase exist: one is bound to the cell wall, and one is liberated by the cell
as “free coagulase.” Slide coagulase test detects the bound coagulase (clumping
factor), which acts directly on the fibrinogen in plasma and causes clumping of
bacteria. Slide coagulase test results agree approximately 96% with tube coagulase
test results. Coagulase-positive organism forms clumps within 10 s, but coagulasenegative
organism remains uniformly suspended.
Using a sterile pipette, a drop of sterile saline is placed on a glass slide. One to two
colonies of the organism is emulsified in the saline and tested for autoagglutination.
A drop of rabbit plasma is placed on the slide and mixed for few seconds and
observed for clumping within 10 s.
A positive slide coagulase test result is valid only for strains of Staphylococcus
spp. that are negative for autoagglutination or stickiness. Coagulase is also present
in S. intermedius and S. hyicus, but these species are infrequent clinical isolates.
Similarly, clomping factor is produced by S. scheiferi and S. lugdunensis and may
give false-positive reactions (Isenberg, 1992; Koneman et al., 1997).
Microdase
Microdase disk is a reagent-impregnated disk used in the differentiation of Staphylococcus
from Micrococcus by the detection of the oxidase enzyme. In the
presence of atmospheric oxygen, the oxidase enzyme reacts with tetramethylp-phenylenediamine
(TMPD) in the disk and cytochrome C in the organism to
form a colored compound. All micrococci contain cytochrome C, whereas most
staphylococci lack cytochrome C. The oxidase reagent substantiates the presence
of type C cytochrome.
Microdase disk is placed on a glass slide and inoculated with several isolated
colonies. The disk is examined for up to 2 min for development of a blue color (positive
reaction). No color change or a white to gray color after 2 min is considered
a negative reaction.