X and V Factor Test: Principle, Procedure, Results

Haemophilus spp. is a genus of Gram-negative, aerobic or facultatively anaerobic, pleomorphic (mostly coccobacilli), Gammaproteobacteria of the phylum Pseudomonadota and family Pasteurellaceae. Several species of the genus Haemophilus are pathogenic to humans like H. influenzae, H. parainfluenzae, H. ducreyi, H. aegyptius, etc.

Haemophilus spp. needs either or both of the two growth factors for their growth and survival, namely, hemin or protoporphyrin IX (X-factor) and nicotinamide adenine dinucleotide (NAD) (V-factor). Based on their need of either one or both of these growth factors Haemophilus spp. can be differentiated to species level. This test is called X and V factor tests.  

Objectives of X and V Factor Test

• To assess the need for the X factor and V factor for growth by Haemophilus spp. 
• To differentiate Haemophilus isolates into different species

Principle of X and V Factor Test

Some species of the genus Haemophilus require either X-factor or V-factor while some require both X and V factors for growth. This significant difference in the requirement of X and V growth factors is the basis of the differentiation of Haemophilus spp. in this test. 

While inoculating and incubating in a general nutrient medium (medium lacking X and V factors);

1.  Those bacteria that require only the X-factor but not the V-factor for survival will grow around X-disk and XV-disk but not around V-disk. 
2. Similarly, those bacteria that require the V-factor only but not the X-factor for survival will grow around the V-disk and XV-disk but not around X-disk. 
3. And, those bacteria that require both X-factor and V-factor will grow around the XV-disk but not around the X-disk and V-disk. 

Requirements for X and V Factor Test

A. Culture Media

A general media without X and V factors is used for performing this test. Trypticase Soy Agar (Soyabean Casein Digest Medium), Blood Agar Base, or Nutrient Agar is suitable for performing this test. 

i. Composition of Soyabean Casein Digest Medium per 1000 mL

Tryptone- 15.00 grams

Soya Peptone- 5.00 grams

Sodium Chloride- 5.00 grams

Agar- 15.0 grams

Final pH- 7.3±0.2 at 25°C

(Reference: https://www.himedialabs.com/media/TD/M290.pdf) 

ii. Preparation of Soyabean Casein Digest Medium

• Measure the appropriate amount of Soyabean Casein Digest media powder (40.0 grams per 1000 mL) and dissolve it in the water of the required volume in a conical flask (or glass bottle). 
• Stir well in a magnetic stirrer or manually and heat to boil to completely dissolve the media powder.
• Autoclave the medium at 121°C and 15 lbs pressure for 15 minutes to sterilize.
• Let the medium cool to about 40 to 45°C and dispense about 20 to 25 mL of medium in each petri plate of 10 cm diameter and allow the medium to solidify completely.

B. Reagents

• X-factor disk
• V-factor disk
• XV-factor disk

C. Equipment

D. Sample Bacteria (Test Bacteria)

E. Control Organism

Haemophilus influenzae ATCC 35056

Haemophilus parainfluenzae ATCC 7901

Haemophilus ducreyi ATCC 33940

Procedure of X and V Factor Test

1. Prepare a heavy inoculum of Haemophilus spp. (equivalent to 0.5 McFarland standard).
2. Using a sterile inoculating loop or swab, uniformly spread the inoculum heavily over the surface of the Soyabean Casein Digest medium plate. 
3. Allow the suspension to diffuse and dry for about 2 to 3 minutes. 
4. Using sterile forceps, place X-disk, V-disk, and XV-disk in the petri plate (at least 40 mm apart from each other). 
5. Incubate the plate at the inverted position in an aerobic incubator or at 3 to 5% CO₂ overnight at 35±2°C.
6. Observe for the growth of bacteria in the vicinity of the disks.

Result and Interpretation of X and V Factor Test

• The development of visible colonies around the X-disk and the XV-disk only indicates the bacterium needs the X-factor for growth. 
• The development of visible colonies around the V-disk and the XV-disk only indicates the bacterium needs the V-factor for growth. 
• The development of visible colonies around the XV-disk only indicates the bacterium needs both the X-factor and the V-factor for growth.

Quality Control

• Haemophilus influenzae ATCC 35056 grows around XV-disk only. 
• Haemophilus parainfluenzae ATCC 7901 grows around V-disk and XV-disk. 
• Haemophilus ducreyi ATCC 33940 grows around X-disk and XV-disk. 

X and V Factor Test Results 

Precautions

• Do not use medium containing X and V factors like Blood agar, chocolate agar, etc. 
• Use heavy inoculum of bacteria and spread uniformly all over the surface of the medium. 
• Place the disks at least 40 to 50 mm apart from each other.
• Incubation in CO₂-rich conditions may give a false positive result for the V-factor. 
• Ensure a completely sterile environment and materials before performing the test otherwise false positive results may be seen. 

Applications of X and V Factor Test

• To classify Haemophilus spp. 
• To identify H. influenzae in a medical lab.

Limitations of X and V Factor Test

• This test has limited application; differentiation of Haemophilus spp. and a few bacteria like Abiotrophia spp. and Granulicatella spp. 
• H. influenzae and H. aegyptius give similar reaction result, so it is very difficult to differentiate them with this test.
• The bacteria first need to be identified as Haemophilus spp. 
• Test results of other biochemical tests and even molecular analysis may be required for complete identification.

References

1. Musher DM. Haemophilus Species. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 30. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8458/
2. An X-factor Requiring Haemophilus Species. By W. J. RYAN. J. gen. Microbiol. (1968)~ 52, 275-286. https://doi.org/10.1099/00221287-52-2-275
3. Morton, D. J., VanWagoner, T. M., Seale, T. W., Whitby, P. W., & Stull, T. L. (2008). Catalase as a source of both X- and V-factor for Haemophilus influenzae. FEMS Microbiology Letters, 279(2), 157-161. https://doi.org/10.1111/j.1574-6968.2007.01020.x
4. Khattak ZE, Anjum F. Haemophilus influenzae Infection. [Updated 2023 Apr 27]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK562176/
5. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/800302/TP_38i4.pdf
6. https://microbiologyinfo.com/x-and-v-factor-test/
7. https://microbeonline.com/x-v-factor-test-haemophilus-principle-procedure-results/
8. https://bioactiva.com/pub/media/sebwite/productdownloads//x/-/x-factor.pdf
9. https://microbiologynote.com/x-and-v-factor-test/
10. https://www.himedialabs.com/media/TD/DD021.pdf
11. https://www.himedialabs.com/media/TD/DD020.pdf
12. https://www.himedialabs.com/media/TD/DD022.pdf
13. https://www.onlinebiologynotes.com/satellitism-test-for-identification-of-haemophilus-influenzae/