X and V factor test for Haemophilus:  Principle, Procedure and Results

X factor (hemin) and V factor (nicotinamide-adenine-dinucleotide, NAD) requirement test is done for the identification and differentiation of  Haemophilus spp. especially identification of Haemophilus influenzae, which causes deadly diseases like meningitis, pneumonia among others.

Haemophilus spp are small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements. H. influenzae is a fastidious organism which grows best at 35-37°C with ~5% CO2 (or in a candle-jar) and requires both X factor and V factor for growth. H. influenzae cannot grow on an unsupplemented Blood Agar Plate (lacks V factor) or any other general purpose medium such as Trypticase Soy Agar, Nutrient Agar (lacks both X and V factor) etc. Haemophilus ducreyi the causative agents of  sexually transmitted infections (genital chancre) requires factor X for its growth whereas Haemophilus parainfluenzae (causative agent of pneumonia and endocarditis) requires only factor V and is able to grow on blood agar.

Mnemonic to remember growth factor requirements of most common pathogenic species of Haemophilus 

Haemophilus parainfluenzae requires factor V (p means pannch=V)
Haemophilus ducreyi requires factor X (ddus means X)
Hamophilus influenzae requires factor X and V

H. influenzae can be grown in a blood agar plate previously streaked with beta-hemmolytic Staphylococcus aureus. As hemolysis by S. aureus liberates V factor, there will growth of the H.influenzae around the streak line, a property known as satellitism. 

Fig. 1: Flow chart for identification and characterization of a H. influenzae isolate

Chocolate Agar (heat-lysed sheep or horse blood agar) plate is the standard medium used for growth of H. influenzae. Growth occurs on a Chocolate Agar because NAD is released from the blood during the heating process of chocolate agar preparation (the heating process also inactivates growth inhibitors) and hemin is available from non-hemolyzed as well as hemolyzed blood cells.

Growth factor requirements can be determined using the principles of agar diffusion with the use of paper disks or strips containing X, V and xv factors.

This strain is presumptively Haemophilus influenzae as it has grown only around the disk containing both XV factor.


  1. Prepare a heavy suspension of cells (1 MacFarland  turbidity standard) of the organism in saline.
    • Note it is important not to carry over any X-factor or V factor contained in the medium that the organism is taken from.
  2. Dip a sterile swab into the organism suspension. Roll the swab over the entire surface of a trypticase soy-agar plate.
  3. Place the X, V , and XV factor disks on the agar surface. If using separate disks, place them at least 4 to 5 cm apart.
  4. Carefully invert the plate and incubate overnight at 35°C in place it in a CO2-incubator or candle extinction jar.

Expected Results

H. influenzae will grow only around the XV disk (i.e., the disk containing both X and V factors).

Growth Around  Inference
X disk V disk XV disk Entire Agar surface
No No Yes No Requirement for both X and V factor
No Yes Yes No Requirement for V factor
Yes Yes Yes Yes No requirement for either X or V factor

Quality Control

  1. Positive:
    1. Haemophilus influenzae will show a halo of growth around XV disk; the rest of the agar surface will show no growth.
    2. Haemophilus parainfluenzae will show a halo of growth around XV and V disks.
  2. Negative
    1. Haemophilus aphrophilus will grow over the entire surface of the plate. Neither X, nor V, nor XV factors are necessary for growth.


H. haemolyticus also shows similar results (only grow around the disk containing both hemin and NAD). Haemophilus ID Quad plates can be performed to differentiate between these two isolates. H. haemolyticus gives β hemolysis when grown on media containing horse blood but H. influenzae grow on such media but does not hemolyze the horse blood cells.

References and further reading: 

  1. Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae (Images and flow chart are taken from this manual)