Advanced Techniques in Diagnostic Microbiology

6. Biochemical Profile-Based Microbial ID Systems 101
salt-tolerant, from non-enterococcal group D streptococci, such as S. bovis and
S. equinus.
Inoculate the tube containing 6.5% sodium chloride with the organism and
incubate at 35 ◦ C in non-CO 2 for 24–48 h. A visible growth (turbidity) is considered
positive and no growth is considered negative.
If the medium is inoculated too heavily, the inoculum may be interpreted as
growth, resulting in a false-positive reaction. Aerococcus, Pediococcus, Staphylococcus,
and up to 80% of group B Streptococcus can grow in 6.5% salt broth. In
addition, Aerococcus may also be bile esculin positive (Isenberg, 1992; Koneman
et al., 1997).
Indole Test
Indole, a benzyl pyrrole, is one of the metabolic degradation products of the amino
acid tryptophan. Bacteria that possess the enzyme tryptophanase are capable of
hydrolyzing and deaminating tryptphan with the production of indole, pyruvic acid,
and ammonia. The indole test is based on the formation of a red color complex
when indole reacts with the aldehyde group of p-dimethylaminobenzaldehyde, the
active chemical in Kovac’s reagent. In order to perform this test, the organism must
be grown on a medium rich in tryptophan such as indole nitrite broth.
Inoculate the indole nitrite broth medium with 2–3 colonies of the organism to be
tested. Incubate the tubes at 35 ◦ C in a non-CO 2 incubator for 24–48 h. Examine the
tubes for growth. When the broth is visibly turbid, use a sterile pipette to transfer
3 mL into a sterile tube. Add 1 mL of xylene to the contents of the tube, which
extracts the indole, if present, from the broth into the xylene. Wait 1–2 min, and
add 0.5 mL Kovac’s reagent and observe for the production of a pink to red color in
the xylene layer. A pink to red color at the interface the of the reagent and the broth
within seconds after the addition of Kovac’s reagent indicates a positive reaction.
No color change indicates a negative reaction (Koneman et al., 1997).
Nitrite Test
Organisms that reduce nitrate have the ability to extract oxygen from nitrates to
form nitrites and other reduction products. The presence of nitrites in the medium
are detected by the formation of a red diazonium dye, p-sulfobenzeneazo-αnaphthylamine,
following the addition of α-naphthylamine and sulfanilic acid.
If no color develops after adding the reagents, this indicates that nitrates have not
been reduced (a true negative reaction) or that they have been reduced beyond the
oxidation level of nitrite to products such as ammonia, nitrogen gas (denitrification),
nitric oxide (NO), or nitrous oxide (N 2 O) and hydroxylamine. Because the
test reagents detect only nitrites, the latter process would lead to a false-negative
result. Therefore, it is necessary to add a small amount of zinc dust to all negative
reactions. Because zinc ions reduce nitrates to nitrites, the development of a red
color after adding zinc dust indicates the presence of nitrates and confirms a true
negative reaction.