X and V Factor Test for Haemophilus- Principle, Procedure, Uses and Results Interpretation

 

Objective

The X and V factor test is used to identify the requirements for the growth of Haemophilus species, specifically the presence of factors X (hemin) and V (nicotinamide adenine dinucleotide, or NAD).

Principle

Haemophilus species require certain growth factors that are not present in regular culture media. The two main factors are X (hemin) and V (NAD). Hemin is required for the synthesis of certain enzymes, and NAD is required for energy metabolism. The X and V factor test determines if a Haemophilus strain requires either or both of these factors for growth. Filter paper strips or disks impregnated with X and V factors are commercially available. X and V factors, each being water-soluble, readily diffuse in agar culture media. Filter paper disks or strips impregnated with these factors are placed on the surface of a medium deficient in these factors, such as trypticase-soy or brain–heart infusion agar, which has been inoculated as a lawn with the test organism. Factor requirements of the organism are then determined, after overnight incubation, by observing the patterns of growth around the paper strips or disks.

Media and Reagents

• Paper strips or disks impregnated with X and V factors
• An agar medium deficient in X and V factors. Trypticase-soy or brain–heart infusion agars are suitable.
• Brain–heart infusion broth

Quality Control

A. Haemophilus parainfluenzae: requires V factor only

B. Haemophilus influenzae: requires both X and V factors

Test Procedure

• From a pure culture of the organism to be identified, prepare a light suspension of the growth in the brain–heart infusion broth. Be careful when transferring the growth not to pick up any of the hemin containing medium from the culture plate. From this suspension, inoculate the surface of the factor deficient agar medium.
• Place an X and a V factor strip on the surface of the agar in the area of inoculation.
• Position the strips about 1 cm apart.
• Incubate the plate in 3%–5% CO2 at 35°C for 18–24 hours.

Results and Interpretation

Visually inspect the agar surface, observing closely for visible growth between or around one or more of the strips or disks. The following patterns indicate the need for X factor only, V factor only, or both X and V factors.

X and V factor test

Limitations of X and V factor test

• False positives: Some bacteria, such as Neisseria species, can produce factor V, which can lead to false positives in the test. This can result in misidentification of the Haemophilus species.
• False negatives: Some strains of Haemophilus may grow without the addition of X or V factors, leading to false negatives. This can result in the failure to identify certain Haemophilus species.
• Variability in factor production: The quality and quantity of X and V factors produced by the Staphylococcus aureus strain used in the test can vary, leading to inconsistent results.
• Limited information: The X and V factor test only provides information on the requirements for growth of Haemophilus species, and does not provide information on other characteristics such as biochemical properties, antibiotic resistance, or virulence factors.
•  Time-consuming: The X and V factor test is a time-consuming procedure that requires at least 24-48 hours of incubation, which can delay the identification of the Haemophilus species.

Reference

• Koneman’s Color Atlas and Text book of Diagnistic Microbiology.
• Bailey and Scott’s Diagnostic Microbiology.
• Parker RH, Hoeprich PD. Disk method for rapid identification of Haemophilus sp. Am J Clin Pathol 1963;37:319–327.