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

The objective of X and V factor test is to differentiate among Haemophilus species based on their requirements for X and V factors for growth. Haemophilus species are gram-negative bacteria that can cause various infections in humans, such as respiratory tract infections, meningitis, otitis media, conjunctivitis, and sexually transmitted diseases. Some Haemophilus species require certain growth factors that are not present in the usual culture media, such as blood agar or chocolate agar. These growth factors are X factor (hemin) and V factor (nicotinamide-adenine-dinucleotide or NAD). The presence or absence of these factors can help identify the species of Haemophilus and guide the appropriate treatment. For example, Haemophilus influenzae requires both X and V factors, Haemophilus parainfluenzae requires only V factor, and Haemophilus ducreyi requires only X factor. Therefore, the X and V factor test is a simple and useful method to distinguish among these clinically important bacteria.

Haemophilus spp. are gram-negative bacteria that have varying requirements for X and V growth factors. X factor indicates hemin, which is a component of hemoglobin that provides iron for the bacteria. V factor indicates nicotinamide-adenine-dinucleotide (NAD), which is a coenzyme that participates in various metabolic reactions.

The presence or absence of these factors in the culture medium can be used to differentiate among Haemophilus species. For example, Haemophilus influenzae requires both X and V factors for growth, while Haemophilus parainfluenzae requires only V factor and Haemophilus ducreyi requires only X factor.

The X and V factor test is based on the diffusion of these factors from impregnated disks or strips into the agar surface where a lawn of the test organism is inoculated. The bacteria will grow only around the disk or strip that provides the appropriate factor or factors for their growth. The growth pattern around the disks or strips can be observed after overnight incubation and compared with the expected results for each species.

The X and V factor test is a simple and reliable method to identify Haemophilus spp. based on their growth factor requirements. However, it should not be the sole criterion for species identification because some species may have similar or overlapping requirements. Other tests, such as biochemical tests, serological tests, or molecular tests, may be needed to confirm the identification of Haemophilus spp.

The procedure of X and V factor test is simple and straightforward. It involves the following steps:

• Make a very light suspension (McFarland 0.5) of the organism in sterile saline. This ensures that the organism is evenly distributed on the agar surface and does not interfere with the diffusion of the growth factors from the disks.
• Dip a sterile swab into the organism suspension. Roll the swab over the entire surface of a trypticase soy agar plate. This medium is suitable for the growth of most Haemophilus species and does not contain any X or V factors.
• 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. The disks are impregnated with hemin (X factor), NAD (V factor), or both (XV factor). The disks provide a source of growth factors for the organisms that require them.
• Incubate the plate overnight at 35°-37°C in ambient air. This temperature and atmosphere are optimal for the growth of Haemophilus species. The incubation time allows for sufficient diffusion of the growth factors from the disks into the surrounding medium.

The procedure is simple and can be performed in any microbiology laboratory with basic equipment and materials. The results can be observed visually after incubation. The procedure can help to differentiate among Haemophilus species based on their X and V factor requirements.

The results of the X and V factor test are based on the presence or absence of growth around the disks that contain the growth factors. The following table summarizes the possible outcomes and interpretations of the test:

The image below shows an example of a positive test for Haemophilus influenzae, which grows only around the XV disk.

The image below shows an example of a negative test for Haemophilus aphrophilus, which grows over the entire surface of the agar.

The X and V factor test is a simple and reliable method to differentiate among Haemophilus species based on their growth factor requirements. It can help in the identification and diagnosis of infections caused by these bacteria.

• The X and V factor test is a simple and rapid method to differentiate among Haemophilus species based on their growth factor requirements. However, this test has some limitations that should be considered before using it as the sole criterion for species identification.
• One limitation is that some Haemophilus species may have overlapping or variable growth factor requirements. For example, some strains of H. influenzae may grow without V factor if they have acquired the ability to synthesize NAD from other sources. Similarly, some strains of H. parainfluenzae may grow without X factor if they have acquired the ability to synthesize hemin from other sources. Therefore, the X and V factor test may not accurately reflect the true growth factor requirements of some Haemophilus strains.
• Another limitation is that some Haemophilus species may have additional growth factors or conditions that are not detected by the X and V factor test. For example, H. ducreyi requires not only X factor but also a high concentration of carbon dioxide (10%) for optimal growth. H. aphrophilus and H. paraphrophilus require not only V factor but also arginine for optimal growth. Therefore, the X and V factor test may not provide sufficient information to distinguish among some Haemophilus species that have similar growth factor requirements but different nutritional or environmental needs.
• A third limitation is that some Haemophilus species may be confused with other bacteria that have similar growth factor requirements or morphology. For example, some strains of Pasteurella multocida may require both X and V factors for growth, mimicking H. influenzae. Some strains of Actinobacillus actinomycetemcomitans may require only V factor for growth, mimicking H. parainfluenzae. Some strains of Neisseria gonorrhoeae may appear as small gram-negative coccobacilli on Gram stain, resembling Haemophilus spp. Therefore, the X and V factor test may not be sufficient to differentiate Haemophilus spp from other bacteria that have similar characteristics.
• In conclusion, the X and V factor test is a useful screening tool to differentiate among Haemophilus species based on their growth factor requirements. However, this test has some limitations that should be taken into account before using it as the sole criterion for species identification. Other tests, such as biochemical reactions, serological typing, molecular methods, or susceptibility testing, may be needed to confirm the identity of Haemophilus spp and to distinguish them from other bacteria that have similar growth factor requirements or morphology.

To ensure the accuracy and reliability of the X and V factor test, it is important to use appropriate quality control strains that have known growth factor requirements. The following strains are recommended for quality control:

• Haemophilus influenzae (ATCC35056): This strain requires both X and V factors for growth. It should show a halo of growth around the XV disk only, and no growth on the rest of the agar surface. This indicates a positive test for both factors.
• Haemophilus parainfluenzae (ATCC7901): This strain requires only V factor for growth. It should show a halo of growth around the XV and V disks, and no growth around the X disk. This indicates a positive test for V factor only.
• Haemophilus ducreyi (ATCC27722): This strain requires only X factor for growth. It should show a halo of growth around the XV and X disks, and no growth around the V disk. This indicates a positive test for X factor only.

If the quality control strains show the expected results, then the test is valid and can be used to differentiate among Haemophilus species based on their growth factor requirements. If the quality control strains do not show the expected results, then the test is invalid and should be repeated with fresh media and disks.

The X and V factor test is a simple and rapid method to differentiate among Haemophilus species based on their characteristic utilization of X and V growth factors. This test is useful for the identification of Haemophilus influenzae, which is a major cause of respiratory infections, meningitis, otitis media, and other diseases in humans. Haemophilus influenzae requires both X and V factors for growth and shows positive growth around the XV disk only.

Other Haemophilus species that can cause human infections include Haemophilus parainfluenzae, Haemophilus ducreyi, Haemophilus aphrophilus, and Haemophilus parahaemolyticus. These species have different growth factor requirements and can be distinguished by the X and V factor test. For example, Haemophilus parainfluenzae requires only V factor and shows positive growth around the XV and V disks. Haemophilus ducreyi requires only X factor and shows positive growth around the XV and X disks. Haemophilus aphrophilus and Haemophilus parahaemolyticus do not require either X or V factor and show positive growth over the entire surface of the agar.

The X and V factor test can also be used to identify some other bacteria that have similar growth factor requirements as Haemophilus spp. For example, Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, requires both X and V factors for growth. Pasteurella multocida, which causes fowl cholera and pasteurellosis in various animals, requires only V factor for growth. The X and V factor test can help differentiate these bacteria from other related genera such as Mannheimia and Bibersteinia.

The X and V factor test is a valuable tool for the diagnosis of bacterial infections caused by Haemophilus spp. and other related bacteria. It can help guide the appropriate treatment and prevention measures for these infections. However, this test should not be the sole criterion for species identification because similarities exist in growth factor requirements of some bacteria. Other biochemical tests, serological tests, or molecular methods should be used to confirm the identification of the bacteria.

1. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology. 12th ed. St. Louis, Missouri: Mosby Elsevier; 2007. p. 357-358.
2. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. p. 244-245.
3. Mahon CR, Lehman DC, Manuselis G. Textbook of Diagnostic Microbiology. 5th ed. St. Louis, Missouri: Elsevier Saunders; 2015. p. 405-406.
4. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 8th ed. Philadelphia: Elsevier Saunders; 2016. p. 260-261.

The X and V factor test is useful for the identification and differentiation of Haemophilus species, which are important pathogens of humans and animals. Some of the clinical applications of this test are:

• Diagnosis of Haemophilus influenzae infections: H. influenzae is a common cause of respiratory tract infections, meningitis, otitis media, sinusitis, and conjunctivitis. It can also cause invasive diseases such as septicemia and endocarditis. H. influenzae requires both X and V factors for growth and shows positive growth around the XV disk only in the X and V factor test.
• Diagnosis of Haemophilus ducreyi infections: H. ducreyi is the causative agent of chancroid, a sexually transmitted disease characterized by painful genital ulcers and lymphadenitis. H. ducreyi requires only X factor for growth and shows positive growth around the XV and X disks in the X and V factor test.
• Diagnosis of Haemophilus parainfluenzae infections: H. parainfluenzae is a normal inhabitant of the upper respiratory tract and can cause opportunistic infections such as endocarditis, bacteremia, meningitis, and respiratory tract infections. H. parainfluenzae requires only V factor for growth and shows positive growth around the XV and V disks in the X and V factor test.

The X and V factor test can also be used for epidemiological purposes to track the prevalence and distribution of Haemophilus species in different regions and populations.

The X and V factor test is a simple and reliable method to differentiate among Haemophilus species based on their characteristic utilization of X and V growth factors. This test is especially useful for the identification of Haemophilus influenzae, which causes deadly diseases like meningitis, pneumonia, otitis media, sinusitis, and septicemia. Haemophilus influenzae is a fastidious organism that requires both X and V factors for growth. Other Haemophilus species have varying requirements for X and V factors. For example, Haemophilus parainfluenzae requires only V factor, Haemophilus ducreyi requires only X factor, and some Haemophilus species do not require either factor.

The X and V factor test can also be used to identify other bacteria that have similar growth factor requirements as Haemophilus spp. For example, some strains of Pasteurella multocida require both X and V factors, while others require only V factor. Some strains of Actinobacillus pleuropneumoniae require only V factor. The X and V factor test can help distinguish these bacteria from other gram-negative bacilli or coccobacilli that do not require X or V factors.

The X and V factor test is a valuable tool for the diagnosis and treatment of infections caused by Haemophilus spp. and other bacteria that depend on X or V factors. By knowing the growth factor requirements of the causative agent, appropriate culture media and antimicrobial agents can be selected for optimal isolation and identification. The X and V factor test can also help monitor the epidemiology and prevalence of different Haemophilus spp. in various populations and regions.

1. Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology. 12th ed. St. Louis, Missouri: Mosby Elsevier; 2007. p. 357-358.
2. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. p. 244-245.
3. Mahon CR, Lehman DC, Manuselis G. Textbook of Diagnostic Microbiology. 5th ed. St. Louis, Missouri: Elsevier Saunders; 2015. p. 405-406.
4. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 8th ed. Philadelphia: Elsevier Saunders; 2016. p. 260-261.

We are Compiling this Section. Thanks for your understanding.