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.

Procedure:

• 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 35-37^o

Expected results:

• 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)

Quality control:

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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