Cat. no. Z7031 X-Factor 1x50 disks/cartridge
Cat. no. Z7041 V-Factor 1x50 disks/cartridge
Cat. no. Z7051 XV-Factor 1x50 disks/cartridge


Hardy Diagnostics X - and V-Factor Disks are paper disks impregnated with X (hemin) and V (nicotinamide adenine dinucleotide - NAD) growth factors. They are used for the differentiation of Haemophilus species, including Aggregatibacter aphrophilus based upon their requirements for the growth factors.


Haemophilus spp. have varying requirements for X and V growth factors. Consequently, the significant differences in growth factor requirements of Haemophilus spp. allows for their differentiation. Differentiation is based on the presence or absence of growth around and/or between disks impregnated with factors X, V and XV.

Use of paper disks impregnated with the growth factors was first proposed by Parker and Hoeprich.(4)

The determination that H. influenzae requires two growth factors was made by Davis, Thjotta, and Avery.(1,2)


Each X-Factor Disk is impregnated with hemin. Each V-Factor Disk is impregnated with NAD (nicotinamide adenine dinucleotide). Each XV-Factor Disk is impregnated with a combination of hemin and NAD.


Storage: Upon receipt, store at -20 to 8ºC. away from direct light. The disks should not be used if there are any signs of deterioration, discoloration, or if the expiration date has passed. Protect from light, excessive heat, and moisture.

The expiration date applies to the product in its intact packaging when stored as directed.



1. Using aseptic techniques, select isolated colonies of suspect colonies and prepare a suspension equivalent to a 0.5 McFarland standard (Cat. no. MS05). Tryptic Soy Broth (Cat. no. R30) or sterile Saline, 0.85% (Cat. no. K59) can be used for making suspensions.

Note: Due to the possibility of carryover of growth factors, do not cool the inoculating loop in the primary isolation medium before selecting colonies.

2. Using a sterile swab, inoculate the entire surface of a factor-free media such as Brain Heart Infusion Agar (Cat. no. W15). If Brain Heart Infusion Agar is unavailable, Mueller Hinton Agar (Cat. no. G45) or Tryptic Soy Agar (Cat. no. G60) may be substituted. Inoculate by streaking in three different directions by rotating the plate in a 60 degree angle after each streaking. Streaking in this manner ensures confluent growth.

3. Allow the surface to dry 3-5 minutes.

4. Using aseptic technique, apply disks around the periphery of the plate (approximately 1-2cm from the edge of the medium). Apply disks in the following positions:

X-Factor Disk 12 o'clock
V-Factor Disk 4 o'clock
XV-Factor Disk 8 o'clock

5. Incubate inoculated media 18-48 hours at 35ºC. in 5-10% CO2. Observe for growth or no growth around the disks.


Organisms that require only X-Factor will grow only in the area of the X- and XV-Factor Disks. Organisms that require only V-Factor, will grow only in the areas of the V- and the XV-Factor Disks. If both X- and V-Factors are required, the organism will grow only in the area of the XV-Factor Disk.

In cases where slight growth occurs around the V-Factor Disk, the growth must be at least equal in amount to that around the XV-Factor Disk before the organism can be considered H. parainfluenzae.

Haemophilus species
Growth Around Disk
Haemophilus aegyptius
Haemophilus aphrophilus
Haemophilus ducreyi
Haemophilus haemolyticus
Haemophilus influenzae
Haemophilus parahaemolyticus
Haemophilus parainfluenzae - + +
Aggregatibacter aphrophilus (formerly H. parainfluenzae, ATCC® 7901)


Because similarities exist in growth factor requirements of Haemophilus species, it is not recommended that this procedure be the sole criterion for species identification.

Care should be taken during inoculation of specimens onto culture media in order to prevent nutrient carryover.


Standard microbiological supplies and equipment such as loops, slides, staining supplies, other culture media, microscopes, incinerators, and incubators, etc., as well as catalase, and other biochemical and serological reagents, are not provided.


Expected Results
Test Organisms
Haemophilus influenzae
ATCC ® 10211
X-Factor: No Growth
V-Factor: No Growth
XV-Factor: Growth

Aggregatibacter aphrophilus (formerly Haemophilus parainfluenzae) ATCC ® 7901 X-Factor: No Growth
V-Factor: Growth
XV-Factor: Growth

User Quality Control

It is recommended that each lot or shipment of XV-Factor disks be checked with a positive control, and the X-Factor and V-Factor disks be tested with both known positive and negative controls.(6,7)

XV-Factor Growth

Showing growth of Haemophilus influenzae (ATCC® 10211) around the XV-Factor Disk (Cat. no. Z1051) only. This growth pattern demonstrated the inability of the organism to grow in the absense of either V-Factor or X-Factor and was consistant with H. influenzae. Incubated on Mueller Hinton Agar (Cat. no. G45) in CO2 for 48 hours at 35ºC.

XV-Factor Growth

Showing closer image of Haemophilus influenzae growth around the XV-Factor disk from the photo to the left.


1. Davis, D.J. 1917. J. Infect. Dis.; 21:392.

2. Thjotta, T., and Avery, O.T. 1921. J. Exper. Med.; 34:97-114.

3. Lwoff, A., and Lwoff, M. 1937. Studies on codehydrogenosis, I. Proc. Roy. Soc. London Ser. B.; 122:352-359.

4. Parker, R.H., and Hoeprich, P.D. 1962. Am. J. Clin. Path.; 37:319-327.

5. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

6. Commission on Laboratory Accreditation, Laboratory Accreditation Program Microbiology Checklist. College of American Pathologists. Rev. 9/30/2004.

7. Centers for Medicare and Medicaid, Appendix C, Survey Procedures and Interpretive Guidelines for Laboratories and Laboratory Services. Subpart K - Quality System for Non-Waived Testing. 493;1200-1265. www.cms.hhs.gov/clia.

ATCC is a registered trademark of the American Type Culture Collection.