Advanced Techniques in Diagnostic Microbiology

102 J. Aslanzadeh
Using a sterile inoculating loop, an indole nitrite broth medium is inoculated
with 2–3 colonies of the organism to be tested and incubated at 35 ◦ C in a non-CO 2
incubator for 24–48 h. When the broth is visibly turbid, 3 mL of the broth culture
is transferred into a sterile tube and 5 drops of N,N-dimethyl-α-naphthylamine
(nitrate reagent A) is added to the broth. Five drops of sulfanilic acid (nitrate
reagent B) is then added to the broth and observed for the production of a pink to
red color within 30 s. If no color change occurs within 30 s, a small amount of zinc
dust is added and the production of a pink to red color within 10 min is looked for
(Koneman et al., 1997).
ALA (Haemophilus influenzae Porphyrin Test)
The porphyrin test is used in the rapid speciation of Haemophilus by separating
those species that require an exogenous source of X factor from those that do not.
Haemophilus species (H. parainfluenzae and H. parahemolyticus) that produce
the enzyme porphobilinogen synthase have the ability to synthesize heme (factor
X) and therefore do not require an exogenous source of factor X for growth.
Porphobilinogen and porphyrin, precursors in heme synthesis, can be detected
in an enzyme substrate inoculated with a porphobilinogen synthase producing
Haemophilus spp. by the addition of modified Ehrlich’s (Kovac’s) reagent or by
examination with a Wood’s lamp.
Suspend a loopful of organism in 0.5 mL of the enzyme substrate. Incubate
at 35 ◦ Cfor4hifthesuspension is heavy or 18–24 h if the suspension is light.
After incubation add an equal volume of modified Ehrlich’s (Kovac’s) reagent and
vortex the mixture. Allow the substrate and reagent to separate. After the addition
of Kovac’s reagent, a red (pink) color will form in the aqueous phase, indicating the
presence of porphobilinogen, and therefore a positive test for Haemophilus spp. not
requiring factor X. Alternatively, a Wood’s lamp can be used to detect fluorescence
in the reagent phase, indicating the presence of porphyrins, also a positive test. No
coloration or fluorescence indicates a factor X dependent Haemophilus spp. and a
negative test (Killian, 1974).
Motility Indole Lysine (MILS)
MILS medium is a semisolid medium useful in the identification of members of
the Enterobacteriaceae, specifically for screening suspicious colonies from stool
cultures for potential pathogens.
It is used to demonstrate motility, indole production, lysine decarboxylase and
deaminase activity, and hydrogen sulfide production. A small amount of agar is
added to the media for demonstration of motility along a stab line of inoculation.
Growth of motile organisms extends out from the line of inoculation, whereas
nonmotile organisms grow along the stab line.
The pH indicator bromcresol purple is used to facilitate detection of decarboxylase
activity. When inoculated with an organism that ferments dextrose, acids are
produced that lower the pH, causing the indicator in the medium to change from
purple to yellow. The acidic pH also stimulates enzyme activity. Organisms that