Advanced Techniques in Diagnostic Microbiology

6. Biochemical Profile-Based Microbial ID Systems 97
a final identification. As a rule, the classical biochemical identification system is
used to identify fastidious or slow-growing organisms in the reference laboratories.
These isolates are by and large rare biotypes that are not part of the commercial
identification system’s database. Table 6.1 depicts the list of biochemical tests that
are commonly used to identify Gram-negative bacilli (Weyant et al., 1996).
Tube Coagulase Test
Tube coagulase test detects free coagulase (liberated by the cell) that acts on
prothrombin to produce a thrombin-like product that then acts as fibrinogen to
form a fibrin clot.
Prepare a heavy suspension of the Staphylococcus colonies in 0.5 mL of water.
Place the suspension into a tube containing rabbit plasma and incubate at 35 ◦ C for
4 h. Examine for the presence of a clot. If negative for a clot, reincubate the tube
and reexamine at 24 h. Any degree of clot formation at 4 h or 24 h is considered
a positive reaction. No clot formation at 24 h is considered negative coagulase
reaction (Koneman et al., 1997; Forbes et al., 2002; Murray et al., 2003).
DNA Hydrolysis
The DNA hydrolysis test detects the presence of enzyme DNase in an organism.
Using this media, DNase-positive coagulase-positive staphylococci are differentiated
from other Staphylococcus spp. The media contains either toluidine blue or
methyl green, which upon hydrolysis of the incorporated DNA turns colorless.
The media is inoculated with the organism and incubated overnight at 35 ◦ C.
The plate is examined for evidence of growth and loss of color (positive reaction).
No color change indicates a negative reaction (Murray et al., 2003).
Vancomycin Disk Test
The vancomycin disk test is performed as a susceptibility procedure to help differentiate
the Gram-positive, catalase-negative cocci. Aerococcus, Gemella, Lactococcus,
Streptococcus, and some enterococci are susceptible to vancomycin.
Leuconostoc, Pediococcus, Lactobacillus, and some enterococci are resistant to
vancomycin.
A 0.5 McFarland suspension of the organism is prepared in sterile saline. Using
a sterile swab, the bacterial suspension is inoculated onto a BAP. A vancomycin
disk is placed in the center of the inoculated plate and incubated at 35 ◦ CinaCO 2
incubator for 18–24 h. The plate is observed for the presence of a zone of inhibition
around the vancomycin disk. Leuconostoc spp., Pediococcus spp., Lactobacillus
spp., and some Enterococcus spp. are resistant to vancomycin with growth to
the edge of the disk ≤9 mm. Aerococcus spp, Gemella spp., Lactococcus spp.,
Streptococcus spp., and some Enterococcus spp. are susceptible to vancomycin
and produce a zone of inhibition ≥12 mm (Koneman et al., 1997; Murray et al.,
2003).