X and V factor test for Haemophilus

Last updated on June 13th, 2021

X factor (hemin) and V factor (nicotinamide-adenine-dinucleotide, NAD) requirement tests are 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 is 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)
Haemophilus influenzae requires factor X and V

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

Flow chart for identification and characterization of a H. influenzae isolate
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 the 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.
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 McFarland 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 diskV diskXV diskEntire Agar surface 
NoNoYesNoRequirement for both X and V factor
NoYesYesNoRequirement for V factor
YesYesYesYesNo requirement for either X or V factor

X and V Factor Requirement

OrganismX FactorV Factor
Haemophilus influenzae++
H. influenzae biotype aegyptius++
H. haemolyticus++
H. parahaemolyticus+
H. parainfluenzae+
H. paraphrophilus+
H. segnis+
H. ducreyi+
H. aphrophilus
x and v factor requirement test
X and V Factor Requirement Test

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 grows 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)
About Acharya Tankeshwar 476 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

1 Comment

  1. Hello Tankeshwar,
    How are you? I was wondering if you can help me with my discoveries on TSA plates fungi killing B. atrophaeus rapidly. This fungi I have isolated from one of the fresh river water in W. Europe. A slimelike ways, whitish or networks, single fuseform tubelike cells from the fungi engulfing bacteria vegetative cells. This fungi grows quickly at 37 degrees C. The ‘zoning effect’ was also visible around bacteria within TSA plate.
    e-mail me: uwna7333@hotmail.com
    Could it be actinomyceae or phytomyxea family?
    Thank you
    Uweis Nassor

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