Performing Hemin and NAD Growth Factor Requirement Test Using Haemophilus ID Quad Plates

Performing hemin and NAD growth factor requirement test using Haemophilus ID Quad plates

Haemophilus ID Quad plates are another method for determining growth requirements o Haemophilus isolates (Figure 6). While they are more expensive than the paper disks or strips, they can test for β-hemolysis (clear) on horse blood and assist in differentiating H. haemolyticus from H. influenzae.

The Quad plate is divided into four compartments. One quadrant includes medium containing hemin only (Figure 6, lower left). The second quadrant includes medium containing NAD only. The third quadrant contains medium that includes both hemin and NAD The fourth quadrant contains heart infusion agar or blood agar base with 5% horse blood (Figure 6, lower right) for detecting hemolysis and differentiating H. haemolyticus from H. influenzae.

Growth factor requirement procedure using Haemophilus ID Quad plates

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 suspension of cells (comparable to a 0.5 McFarland standard) from overnight growth of suspected Haemophilus on a CAP in trypticase soy broth or distilled water and mix well using a vortex.

3. Use a sterile, inoculating loop to streak one loopful of the cell suspension onto one quadrant of the Quad plate. Streak the entire quadrant, starting at the periphery of the plate and streaking toward the center of the plate.

· Use a different inoculating loop to streak each of the other quadrants with the cell suspension.

· Stab into the blood agar for detection of β-hemolysis (clear).

4. Incubate for 18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).

5. After incubation, examine the individual quadrants for growth and the quadrant with horse blood for hemolysis where the plate was stabbed with the loop.

Reading the Haemophilus ID Quad plate results

H. influenzae will only grow on the quadrant containing both hemin and NAD and the quadrant containing horse blood. It will not hemolyze the horse blood cells.

H. haemolyticus will only grow on the quadrant containing both hemin and NAD and the quadrant containing horse blood. It will hemolyze the horse blood cells.

o H. haemolyticus may lose their hemolytic property when passedin vitro. This has made the definitive identification of H. influenzae and H. haemolyticus using only biochemical tests very difficult and other methods, such as molecular testing, may be employed for differentiating between the two species.

· An organism growing on either the hemin or the NAD quadrant is likely another Haemophilus Species

· If growth occurs on every quadrant, the isolate is probably not a Haemophilus spp.

H. influenzae may occasionally show slight growth in the quadrant containing NAD only.

Quality control of Quad plates

QC should be performed on each new lot of Quad plates before they are used for unknown isolates to ensure that they will support the proper growth of Haemophilus spp. Three plates from each new lot received should be tested using a well-characterized reference strain of H. influenzae , H. haemolyticus, and H. parahaemolyticus. One uninoculated plate from each new lot should also be tested in order to check for contamination of mold or other organisms in the laboratory and/or incubator. QC should be repeated on plates from a lot if they have been exposed to temperatures above 4oC or if there is reason to suspect that the plates have been contaminated since the initial QC was performed.

Procedure for quality control of Quad plates

1. Examine the Quad plates for evidence of microbial contamination, discoloration, drying, deterioration, or other physical defects that may interfere with use. Note the firmness of the agar during the inoculation procedure.

2. Grow the reference strains to be tested for 18-24 hours on a CAP at 35-37°C with ~5% CO2
(or in a candle-jar).

3. Inoculate the Quad plates using cell suspensions from the reference strains as described in the
Quad plate procedure above.

4. Incubate the plates for 18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).

5. As a negative control for contamination, incubate an uninoculated plate from each new lot for 18-24 hours at 35-37°C with ~5% CO2 (or in a candle-jar).

6. Examine the inoculated and uninoculated plates after 18-24 hours for proper growth of Haemophilus spp.

Reading the quality control test results

H. influenzae will only grow on the quadrant containing both hemin and NAD and the quadrant containing horse blood. It will not hemolyze the horse blood cells.

H. haemolyticus will only grow on the quadrant containing both hemin and NAD and the quadrant containing horse blood. It will hemolyze the horse blood cells.
H. parahaemolyticus will grow on all quadrants except for the one containing only hemin. It will hemolyze the horse blood cells.

· Passing result: proper growth of the reference strain on appropriate quadrants and no growth on uninoculated media.

· Failing result: no growth or poor growth of the reference strain on appropriate media and/or growth of organisms on the uninoculated media.