Members of the genus Haemophilus are fastidious in nature. i.e. they require extra nutritional supplements in order to grow. Some Hemophilus require only X factor (haemin-which is present on blood agar) or only V factor (Nicotinamide-adenine dinucleotide, NAD- which is present within the blood cells) or a combination of both. So, Haemophilus influenzae grows only on heated blood agar (Chocolate agar) as it contains both X and V factor as a result of lysis of blood due to heat.
- Prepare a light suspension of the organism isolated in a sterile saline and adjust the turbidity of the suspension with MacFarland 0.5.
- Dip a sterile cotton swab in the suspension
- Roll the swab over entire surface of Muller Hinton agar or Trypticase soy agar plate.
- Place the X, V and XV factor disks on the agar surface. Place them at least 5cm apart.
- Incubate the agar plate overnight at 35-37o
- Growth around XV only – Haemophilus influenzae, Haemophilus haemolyticus (requires both X and V factors)
- Growth around X only – Haemophilus ducreyi (requires X only)
- Growth around V only – Haemophilus parainfluenzae, Haemophilus parahaemolyticus and Haemophilus paraphrophilus
- Growth all over the agar plate – Haemophilus aphrophilus (does not require X and V factors)
Positive Control: Haemophilus influenzae will show a halo of growth around XV disk; the rest of the agar will show no growth. Haemophilus parainfluenzae will show a halo growth around the XV and X disks.
Negative Control: Haemophilus aphrophilus will grow over the entire plate. Neither X nor V nor XV factors are needed for growth.
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