X and V Factor Test: How to Read the Disk Pattern and Avoid False Haemophilus IDs

X factor (hemin) and V factor (nicotinamide-adenine-dinucleotide, NAD) requirement tests are done to identify and differentiate Haemophilus spp. especially identification of Haemophilus influenzae, which causes deadly diseases like meningitis, and pneumonia, among others.

On an unlysed blood agar plate, neither factor is usable. Hemin (X factor) stays locked inside intact red cells, and the NAD (V factor) that is present is destroyed by NADase enzymes released from the red cells and other blood components. This is why H. influenzae, which needs both factors, cannot grow on plain blood agar. Two things unlock the plate: lysis of the red cells (by heat, as in chocolate agar, or by a neighboring Staphylococcus streak) releases hemin, and heat additionally inactivates the NADase so that NAD survives. This single mechanism explains chocolate agar, satellitism, and the disk test that follows.

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. H. influenzae cannot grow on an unsupplemented blood agar plate or any general-purpose medium such as Trypticase Soy Agar or Nutrient Agar. Blood agar holds both factors, but in a form the organism cannot use: hemin trapped inside intact red cells and NAD degraded by red-cell NADase. General-purpose media lack both factors entirely.

Haemophilus ducreyi, the causative agent of sexually transmitted infections (genital chancre), requires factor X for its growth. In contrast, Haemophilus parainfluenzae (causative agent of pneumonia and endocarditis) requires only factor V and can grow on blood agar.

The same mechanism explains satellitism: a Staphylococcus aureus streak on blood agar lyses nearby red cells (freeing hemin) and secretes NAD, so tiny Haemophilus colonies grow only next to the streak. The satellitism test is covered in this article: Satellitism test for Haemophilus influenzae.

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

Chocolate agar is the standard primary medium for growing H. influenzae, because gentle heating lyses the red cells (releasing hemin) and inactivates the NADase (sparing NAD). Its composition, preparation, and colony morphology are covered in the dedicated article: Chocolate agar: composition, preparation, and uses.

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

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

Procedure:

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. Invert the plate and incubate overnight at 35°C 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).

X and VFactorRequirement

Figure: X and V Factor Requirement Test

Quality Control

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

NOTE

H. haemolyticus also shows similar results (only grows around the disk containing hemin and NAD). HaemophilusID 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.

Where Students Get Confused

1. Carryover is the number one cause of a wrong answer. If you pick your test colony straight off chocolate agar and the medium travels with it, you carry hemin and NAD onto the disk plate. The organism then grows all over the plate (or around the wrong disk), and an X- and V-dependent H. influenzae gets misread as needing nothing. Always subculture or wash the inoculum, and never flood the plate with a heavy loopful of chocolate agar growth. This one error explains most "H. influenzae grew everywhere" results.

2. The disk test is unreliable for X (hemin) dependence. Use the porphyrin test. Trace hemin contaminates most basal media, so an X-dependent strain may still grow faintly around the V disk and get called V-only. The more reliable way to prove X dependence is the porphyrin (ALA) test: X-dependent Haemophilus cannot convert delta-aminolevulinic acid (ALA) into porphyrins, so a negative porphyrin test confirms X requirement. If your disk result for X looks borderline, trust the porphyrin test over the disk.

3. "Grows around XV only" is presumptive, not confirmatory. H. haemolyticus gives the identical XV-only pattern as H. influenzae. The two are separated by hemolysis on horse blood agar (H. haemolyticus is beta-hemolytic; H. influenzae is not), not by the disk test. Reporting H. influenzae from the disk pattern alone is a classic exam trap.

4. V is heat-labile, X is heat-stable, and this predicts everything. NAD (V) is destroyed by heat and by RBC enzymes; hemin (X) survives both. That single fact tells you why chocolate agar works (heat kills the NADase), why plain blood agar fails (NADase intact), and why X-dependence is the harder one to demonstrate cleanly.

How to Remember

Species requirements:

• H. parainfluenzae needs V (p = paanch, Nepali for five = V in Roman numerals)
• H. ducreyi needs X (d = das, ten = X in Roman numerals)
• H. influenzae needs both X and V

Heat tells you which factor is fragile: V is the Vulnerable one (heat-labile NAD). X (hemin) is tough and heat-stable. If a question asks which factor survives autoclaving or heating, it is always X.

Reading the plate: growth hugs only the disk that supplies what the organism is missing. Growth around XV only = needs both = think H. influenzae (then confirm with hemolysis).

Key exam facts in one table

References and further reading:

1. Tille P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
2. Leber A. L. (Ed.). (2016). Clinical Microbiology Procedures Handbook (4th ed.). ASM Press.
3. Procop G. W. et al. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
4. World Health Organization. Laboratory Methods for the Diagnosis of Meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. (Source of the identification flow chart.)
5. 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/