X and V Factor Test- Principle, Procedure, Results, Limitations

Objective of X and V Factor Test

To differentiate among Haemophilus species based on X and V factors required for growth.

Principle of X and V Factor Test

Haemophilus spp. has varying requirements for X and V growth factors. X factor indicates hemin and V factor indicates nicotinamide-adenine-dinucleotide, NAD. Consequently, the significant differences in growth factor requirements of Haemophilus spp. allows for their differentiation. Haemophilus spp is small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements. H. influenzae is a fastidious organism that grows best at 35-37°C with ~5% CO2 (or in a candle-jar) and requires both X factor and V factor for growth.  Haemophilus parainfluenzae requires a V factor only for growth while Haemophilus ducreyi requires only X factor without the need of a V factor. H. influenzae can be grown in a blood agar plate previously streaked with beta-hemolytic Staphylococcus aureus. The liberation of the V factor by hemolysis of S. aureus contributes to the growth of the H.influenzae around the streak line. This phenomenon or property is known as satellitism.

For the differentiation of Haemophilus species, a lawn of the test organism is streaked onto heart infusion agar, tryptic soy agar, Haemophilus agar, or nutrient agar. The impregnated disks or strips (X, V, or XV) are placed directly on the confluent inoculation, allowing diffusion of the accessory growth factor into the medium. The organisms will grow only around the disk that provides the appropriate factor for the growth of the organism.

Procedure of X and V Factor Test

  1. Make a very light suspension (McFarland 0.5) of the organism in sterile saline.
  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. Incubate the plate overnight at 35°-37°C in ambient air.
  5. Following incubation examine the plate for growth around the disk.

Result Interpretation of X and V Factor Test

Image Source: Microbe Online and Heungsup Sung.

Positive test:

  • Growth around the XV disk only shows a requirement for both factors.
  • Growth around the V disk, no growth around the X disk, and light growth around the XV disk shows a V factor requirement.

Negative test: Growth over the entire surface of the agar indicates no requirement for either X or V factor.

Limitations of X and V Factor Test

  • It is not recommended that this procedure be the sole criterion for species identification because similarities exist in growth factor requirements of Haemophilus 
  • Care should be taken during inoculation of specimens onto culture media in order to prevent nutrient carryover.

Quality Control of X and V Factor Test

Haemophilus influenza (ATCC35056): halo of growth around the XV disk, no growth on the rest of the agar surface

Haemophilus parainfluenzae (ATCC7901): halo of growth around the XV and V disks

Haemophilus ducreyi (ATCC27722): halo of growth around the XV and X disks


  1. Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen edition, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
  2. UK Standards for Microbiology Investigations. 2015. X and V Factor Test. http://www.apsi.it/public/ufiles/smi/tp38_3_en_150108.pdf
  3. Cooke F.J and Slack M.P.E. 2017. Gram Negative Coccobacilli. https://doi.org/10.1016/B978-0-7020-6285-8.00183-0
  4. Slack M.P.E. 2012. Haemophilus: Respiratory infections; meningitis; chancroid https://doi.org/10.1016/B978-0-7020-4089-4.00046-9
  5. http://himedialabs.com/TD/DD022.pdf

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