Identification of Hemin and NAD as Growth Requirements
H. influenzae
is a fastidious organism and can be identified on the basis of growth
requirements for hemin and NAD. H. influenzae can be differentiated from most
other species of Haemophilus by its specific requirement for both
hemin and NAD for growth H. haemolyticus is the only other species
requiring both hemin and NAD for growth; however, this species
differs from H. influenzae by producing beta-hemolysis (clear) on
horse or rabbit blood.
For patients with bacterial meningitis, H. influenzae must be
considered as the presumptive causative agent as opposed to H. haemolyticus when both hemin and NAD factors are required for growth. To differentiate between the two species, hemolysis must be
checked on horse or rabbit blood agar (see Section II.B., Haemophilus ID Quad plate
section below). H. haemolyticus usually causes hemolysis on these
media, while H. influenzae does not.
It has recently been
reported that H. haemolyticus tend to rapidly lose their hemolytic
property when passed in vitro (2). This has made the
definitive identification of H. influenzae and H.mhaemolyticus using only biochemical tests very difficult and
other methods, such as molecular testing, may be employed for
differentiating between the two species.
Table 1.
Identification of Haemophilus spp. by their growth requirements
for hemin (X factor) and NAD (V factor) and β-hemolysis on horse blood agar
Performing hemin and NAD growth factor requirement test using paper disks and/or strips Growth factor requirements can be identified with paper disks and/or strips using the principles of agar diffusion.
Growth factor requirement procedure using paper disks and/or strips
1. Grow the isolate(s) to be tested for 18-24 hours on a CAP at 35-37°C with ~5% CO2 (or in a candle-jar).
2. Prepare a moderately heavy suspension of cells (comparable to a 1.0 McFarland standard) from overnight growth on the CAP in a suitable broth (trypticase soy, heart infusion, or peptone water) and mix well using a vortex.
Do not transfer any of the chocolate agar media from the plate to the cell suspension as even the smallest amount of agar will affect the test and may lead to misidentification of the bacteria.
3. Inoculate one half of a heart infusion or trypticase soy agar plate with 10 μl of the cell suspension using a sterile loop or swab and allow the suspension to dry.
Two different isolates can be tested on the same plate, but care must be taken to ensure that the cultures do not overlap.
4. Place paper disks or strips containing hemin, NAD, and hemin/NAD on the inoculated plate after the inoculum has dried.
When two bacterial strains are tested on the same plate, the disks must be placed in the exact manner shown, keeping the individual hemin and NAD disks separated by the one containing both factors and leaving as much space between the disks as possible.
5. Perform steps 1-4 using a H. influenzae and a different Haemophilus spp. QC strain to ensure that the hemin and NAD disks or strips are working properly.
6. Carefully invert the plate and incubate for 18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).
7. Observe growth around the paper disks or strips.
Reading the hemin and NAD paper disk and/or strip results
H. influenzae will only grow around the paper disk containing both hemin and NAD, as shown in Figure 5 on the upper half of the plate (see black arrow).
H. haemolyticus will also only grow around the paper disk containing both hemin and NAD. To differentiate between the two species, hemolysis must be checked on horse or rabbit blood agar by inoculating the cell suspension mentioned above on heart infusion agar with 5% rabbit blood (or agar infusion base containing horse blood). Alternatively, a Haemophilus ID Quad plate can be used .
Other Haemophilus spp. will grow around the disk containing both hemin and NAD and either the individual hemin or the NAD disk.
Alternatively, the porphyrin test (2) can be used. This determines the hemin requirement of the isolate while avoiding the problem of hemin carryover from primary culture media and hemin contamination of test media (3).
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